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• Perez-Canadillas JM, Varani G
• Recent advances in RNA-protein recognition.
• Curr Opin Struct Biol. 2001; 11: 53-8
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• The past few years have witnessed remarkable progress in knowledge of the structure and function of RNA-binding proteins and their RNA complexes. X-ray crystallography and NMR spectroscopy have provided structures for all major classes of RNA-binding proteins, both alone and complexed with RNA. New computational and experimental tools have provided unprecedented insight into the molecular basis of RNA recognition.

• Manival X, Ghisolfi-Nieto L, Joseph G, Bouvet P, Erard M
• RNA-binding strategies common to cold-shock domain- and RNA recognition motif-containing proteins.
• Nucleic Acids Res. 2001; 29: 2223-33
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• Numerous RNA-binding proteins have modular structures, comprising one or several copies of a selective RNA-binding domain generally coupled to an auxiliary domain that binds RNA non-specifically. We have built and compared homology-based models of the cold-shock domain (CSD) of the Xenopus protein, FRGY2, and of the third RNA recognition motif (RRM) of the ubiquitous nucleolar protein, nucleolin. Our model of the CSD(FRG)-RNA complex constitutes the first prediction of the three-dimensional structure of a CSD-RNA complex and is consistent with the hypothesis of a convergent evolution of CSD and RRM towards a related single-stranded RNA-binding surface. Circular dichroism spectroscopy studies have revealed that these RNA-binding domains are capable of orchestrating similar types of RNA conformational change. Our results further show that the respective auxiliary domains, despite their lack of sequence homology, are functionally equivalent and indispensable for modulating the properties of the specific RNA-binding domains. A comparative analysis of FRGY2 and nucleolin C-terminal domains has revealed common structural features representing the signature of a particular type of auxiliary domain, which has co-evolved with the CSD and the RRM.

• Wei RR, Richardson JP
• Identification of an RNA-binding Site in the ATP binding domain of Escherichia coli Rho by H2O2/Fe-EDTA cleavage protection studies.
• J Biol Chem. 2001; 276: 28380-7
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• Transcription factor Rho is a ring-shaped, homohexameric protein that causes transcript termination through actions on nascent RNAs that are coupled to ATP hydrolysis. The Rho polypeptide has a distinct RNA binding domain of known structure as well as an ATP binding domain for which a structure has been proposed based on homology modeling. Treatment of Rho with H2O2 in the presence of Fe-EDTA caused single-cut cleavage at a number of points that coincide with solvent-exposed loops in both the known and predicted structures, thereby providing support for the validity of the tertiary and quaternary structural models of Rho. The binding of ATP caused one distinct change in the cleavage pattern, a strong protection at a cleavage point in the P-loop of the ATP binding domain. Binding of RNA and single-stranded DNA (poly(dC)) caused strong protection at several accessible parts of the oligosaccharide/oligonucleotide binding (OB) fold in the RNA binding domain. RNA molecules but not DNA molecules also caused a strong, ATP-dependent protection at a cleavage site in the predicted Q-loop of the ATP binding domain. These results suggest that Rho has two distinct binding sites for RNA. Besides the site composed of multiples of the RNA binding domain, to which single-stranded DNA as well as RNA can bind, it has a separate, RNA-specific site on the Q-loop in the ATP binding domain. In the proposed quaternary structure of Rho, the Q-loops from the six subunits form the upper entrance to the hole in the ring-shaped hexamer through which the nascent transcript is translocated by actions coupled to ATP hydrolyses.

• Burgess BR, Richardson JP
• RNA passes through the hole of the protein hexamer in the complex with the Escherichia coli Rho factor.
• J Biol Chem. 2001; 276: 4182-9
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• Escherichia coli transcription termination factor Rho is a ring-shaped hexameric protein that uses the energy derived from ATP hydrolysis to dissociate RNA transcripts from the ternary elongation complex. To test a current model for the interaction of Rho with RNA, three derivatives of Rho were made containing single cysteine residues and modified with a photo-activable cross-linker. The positions for the cysteines were: 1) in part of the primary RNA-binding site in the N terminus (Cys-82 Rho); 2) in a connecting polypeptide proposed to be on the outside of the hexamer (Cys-153 Rho); and 3) near the proposed secondary RNA-binding site in the ATP-binding domain (Cys-325 Rho). Results from the cross-linking of the modified Rho proteins to a series of lambda cro RNA derivatives showed that Cys-82 Rho formed cross-links with all transcripts containing the Rho utilization (rut) site, that Cys-325 Rho formed cross-links to transcripts that had the rut site and 10 or more residues 3' of the rut site, and that Cys-153 did not form cross-links with any of the transcripts. From a model of the quaternary structure of Rho, which is largely based on homology to the F(1)-ATPase, amino acid 82 is located near the top of the hexamer, and amino acid 325 is located on a solvent-accessible loop in the center of the hexamer. These data are consistent with binding of the rut region of RNA around the crown, with its 3'-segment passing through the center of the Rho hexamer.

• Yamanaka K, Inouye M
• Selective mRNA degradation by polynucleotide phosphorylase in cold shock adaptation in Escherichia coli.
• J Bacteriol. 2001; 183: 2808-16
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• Upon cold shock, Escherichia coli cell growth transiently stops. During this acclimation phase, specific cold shock proteins (CSPs) are highly induced. At the end of the acclimation phase, their synthesis is reduced to new basal levels, while the non-cold shock protein synthesis is resumed, resulting in cell growth reinitiation. Here, we report that polynucleotide phosphorylase (PNPase) is required to repress CSP production at the end of the acclimation phase. A pnp mutant, upon cold shock, maintained a high level of CSPs even after 24 h. PNPase was found to be essential for selective degradation of CSP mRNAs at 15 degrees C. In a poly(A) polymerase mutant and a CsdA RNA helicase mutant, CSP expression upon cold shock was significantly prolonged, indicating that PNPase in concert with poly(A) polymerase and CsdA RNA helicase plays a critical role in cold shock adaptation.

• Worbs M, Bourenkov GP, Bartunik HD, Huber R, Wahl MC
• An extended RNA binding surface through arrayed S1 and KH domains in transcription factor NusA.
• Mol Cell. 2001; 7: 1177-89
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• The crystal structure of Thermotoga maritima NusA, a transcription factor involved in pausing, termination, and antitermination processes, reveals a four-domain, rod-shaped molecule. An N-terminal alpha/beta portion, a five-stranded beta-barrel (S1 domain), and two K-homology (KH) modules create a continuous spine of positive electrostatic potential, suitable for nonspecific mRNA attraction. Homology models suggest how, in addition, specific mRNA regulatory sequences can be recognized by the S1 and KH motifs. An arrangement of multiple S1 and KH domains mediated by highly conserved residues is seen, creating an extended RNA binding surface, a paradigm for other proteins with similar domain arrays. Structural and mutational analyses indicate that the motifs cooperate, modulating strength and specificity of RNA binding.

• van Tilbeurgh H, Le Coq D, Declerck N
• Crystal structure of an activated form of the PTS regulation domain from the LicT transcriptional antiterminator.
• EMBO J. 2001; 20: 3789-99
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• The transcriptional antiterminator protein LicT regulates the expression of Bacillus subtilis operons involved in beta-glucoside metabolism. It belongs to a newly characterized family of bacterial regulators whose activity is controlled by the phosphoenolpyruvate:sugar phosphotransferase system (PTS). LicT contains an N-terminal RNA-binding domain (56 residues), and a PTS regulation domain (PRD, 221 residues) that is phosphorylated on conserved histidines in response to substrate availability. Replacement of both His207 and His269 with a negatively charged residue (aspartic acid) led to a highly active LicT variant that no longer responds to either induction or catabolite repression signals from the PTS. In contrast to wild type, the activated mutant form of the LicT regulatory domain crystallized easily and provided the first structure of a PRD, determined at 1.55 A resolution. The structure is a homodimer, each monomer containing two analogous alpha-helical domains. The phosphorylation sites are totally buried at the dimer interface and hence inaccessible to phosphorylating partners. The structure suggests important tertiary and quaternary rearrangements upon LicT activation, which could be communicated from the protein C-terminal end up to the RNA-binding domain.

• Vaughn JL, Feher VA, Bracken C, Cavanagh J
• The DNA-binding domain in the Bacillus subtilis transition-state regulator AbrB employs significant motion for promiscuous DNA recognition.
• J Mol Biol. 2001; 305: 429-39
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• AbrB is a Bacillus subtilis protein responsible for regulating a diverse array of unrelated genes during periods of sub-optimal growth conditions. DNA binding by AbrB is unique in that sequence recognition is specific, yet no obvious consensus sequence of bound promoter regions is apparent. The N-terminal domain is a recently characterized representative of a novel class of DNA-binding proteins that possess a looped-hinge helix DNA-binding topology. Although the structural characterization of this DNA-binding topology contributed to an understanding of the architectural basis for recognition of DNA target sequences, specific mechanisms responsible for promiscuity in DNA sequence recognition still were not apparent. Analysis of (15)N backbone relaxation parameters shows that dynamic motion of regions directly linked to DNA binding show concerted motion on the microsecond-millisecond timescale. Furthermore, dynamic motion of the hinge region suggests that the DNA-binding region is capable of conformational orientations that allow it to accommodate DNA sequence variability in the cognate binding sites. Copyright 2001 Academic Press.

• Lopez MM, Yutani K, Makhatadze GI
• Interactions of the cold shock protein CspB from Bacillus subtilis with single-stranded DNA. Importance of the T base content and position within the template.
• J Biol Chem. 2001; 276: 15511-8
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• The cold shock protein CspB from Bacillus subtilis binds T-based single-stranded DNA (ssDNA) with high affinity (Lopez, M. M., Yutani, K., and Makhatadze, G. I. (1999) J. Biol. Chem. 274, 33601-33608). In this paper we report the results of CspB interactions with non-homogeneous ssDNA templates containing continuous and non-continuous stretches of T bases. The analysis of CspB-ssDNA interactions was performed using fluorescence spectroscopy, analytical centrifugation and isothermal titration calorimetry. We show that (i) there is a strong correlation between the CspB affinity and stoichiometry and the T content in the oligonucleotide that is independent of which other bases are incorporated into the sequence of ssDNA; (ii) the binding properties of CspB to ssDNA templates with continuous or non-continuous stretches of T bases with similar T content is very similar, and (iii) the mechanism of interaction between CspB and the T-based non-homogeneous ssDNA is mainly through the bases (a stretch of three T bases located in the middle of the ssDNA templates makes the binding independent of the ionic strength). The biological relevance of these results to the role of CspB as an RNA chaperone is discussed.

• Stitt BL
• Escherichia coli transcription termination factor Rho binds and hydrolyzes ATP using a single class of three sites.
• Biochemistry. 2001; 40: 2276-81
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• Escherichia coli transcription termination factor Rho uses the energy of ATP hydrolysis to travel 5' --> 3' along RNA. We previously showed that the hexameric Rho protein binds three molecules of ATP in active sites and that hydrolysis of the three bound ATP molecules upon RNA binding is sequential. Other models of Rho ATP hydrolysis activity have arisen from reports of additional ATP binding sites on Rho. Here we present further evidence from binding, isotope partitioning, and rapid mix/chemical quench experiments, in support of the presence of only three equivalent ATP binding sites on Rho that are catalytic sites and that fire sequentially. These results are incorporated into a proposed mechanism for directional Rho tracking along RNA.

• Kim DE, Patel SS
• The kinetic pathway of RNA binding to the Escherichia coli transcription termination factor Rho.
• J Biol Chem. 2001; 276: 13902-10
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• The Escherichia coli transcription termination factor Rho is structurally and functionally homologous to hexameric helicases that assemble into ring structures. Using stopped-flow fluorescence and presteady-state ATPase kinetics, we have determined the kinetic pathway of poly(C) RNA binding to Rho hexamer, both in the presence and in absence of ATP. These studies indicate a four-step sequential mechanism of RNA binding and reveal the respective roles of the primary and secondary RNA binding sites in initiation and ATPase activation of Rho. The primary RNA binding sites of Rho hexamer interact with poly(C) RNA at a diffusion-limited rate constant close to 8 x 10(8) m(-1) s(-1), resulting in the Rho-RNA species PR1, which subsequently isomerizes to PR2 with a rate constant 21 s(-1). The PR2 isomerizes to PR3 with a rate constant of 32 s(-1) in the presence of ATP, and the formation of PR4 from PR3 results in a species that is fully competent in hydrolyzing ATP at the RNA-stimulated rate. The PR3 to PR4 isomerization occurs at a relatively slow rate of 4.1 s(-1); thus, the presteady-state ATPase kinetics show a distinct lag due to the slow initiation step. The interactions of the RNA with the primary sites trigger ring opening, and we propose that during the last two steps, the RNA migrates into the central channel and interacts with the secondary sites, resulting in the activation of the ATPase activity. The primary RNA binding sites, in addition to promoting sequence specific initiation, kinetically facilitate loading of the RNA into the secondary sites, which are relatively inaccessible, since they are present in the central channel. These studies reveal common features used by hexameric helicases to bind nucleic acids in an efficient and specific manner.

• Izumi H et al.
• Y box-binding protein-1 binds preferentially to single-stranded nucleic acids and exhibits 3'-->5' exonuclease activity.
• Nucleic Acids Res. 2001; 29: 1200-7
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• We have previously shown that Y box-binding protein-1 (YB-1) binds preferentially to cisplatin-modified Y box sequences. Based on structural and biochemical data, we predicted that this protein binds single-stranded nucleic acids. In the present study we confirmed the prediction and also discovered some unexpected functional features of YB-1. We found that the cold shock domain of the protein is necessary but not sufficient for double-stranded DNA binding while the C-tail domain interacts with both single-stranded DNA and RNA independently of the cold shock domain. In an in vitro translation system the C-tail domain of the protein inhibited translation but the cold shock domain did not. Both in vitro pull-down and in vivo co-immunoprecipitation assays revealed that YB-1 can form a homodimer. Deletion analysis mapped the C-tail domain of the protein as the region of homodimerization. We also characterized an intrinsic 3'-->5' DNA exonuclease activity of the protein. The region between residues 51 and 205 of its 324-amino acid extent is required for full exonuclease activity. Our findings suggest that YB-1 functions in regulating DNA/RNA transactions and that these actions involve different domains.

• Hopf M, Gohring W, Ries A, Timpl R, Hohenester E
• Crystal structure and mutational analysis of a perlecan-binding fragment of nidogen-1.
• Nat Struct Biol. 2001; 8: 634-40
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• Nidogen, an invariant component of basement membranes, is a multifunctional protein that interacts with most other major basement membrane proteins. Here, we report the crystal structure of the mouse nidogen-1 G2 fragment, which contains binding sites for collagen IV and perlecan. The structure is composed of an EGF-like domain and an 11-stranded beta-barrel with a central helix. The beta-barrel domain has unexpected similarity to green fluorescent protein. A large surface patch on the beta-barrel is strikingly conserved in all metazoan nidogens. Site-directed mutagenesis demonstrates that the conserved residues are involved in perlecan binding.

• Muto Y, Oubridge C, Nagai K
• RNA-binding proteins: TRAPping RNA bases.
• Curr Biol. 2000; 10: 1921-1921
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• In Bacillus subtilis, tryptophan biosynthesis is regulated by a mechanism called attenuation. The new crystal structure of the 'trp RNA binding attenuation protein', TRAP, in complex with RNA has provided new structural insights into how proteins can bind RNA to regulate transcription and translation.

• Staker BL, Korber P, Bardwell JC, Saper MA
• Structure of Hsp15 reveals a novel RNA-binding motif.
• EMBO J. 2000; 19: 749-57
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• We have solved the crystal structure of the heat shock protein Hsp15, a newly isolated and very highly inducible heat shock protein that binds the ribosome. Comparison of its structure with those of two RNA-binding proteins, ribosomal protein S4 and threonyl-tRNA synthetase, reveals a novel RNA-binding motif. This newly recognized motif is remarkably common, present in at least eight different protein families that bind RNA. The motif's surface is populated by conserved, charged residues that define a likely RNA-binding site. An intriguing pattern emerges: stress proteins, ribosomal proteins and tRNA synthetases repeatedly share a conserved motif. This may imply a hitherto unrecognized functional similarity between these three protein classes.

• Morita EH et al.
• NMR backbone assignments of the cold-regulated RNA-binding protein, rbpA1, in the cyanobacterium, anabaena variabilis M3.
• J Biomol NMR. 2000; 17: 351-2

• Batey RT, Rambo RP, Lucast L, Rha B, Doudna JA
• Crystal structure of the ribonucleoprotein core of the signal recognition particle.
• Science. 2000; 287: 1232-9
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• The signal recognition particle (SRP), a protein-RNA complex conserved in all three kingdoms of life, recognizes and transports specific proteins to cellular membranes for insertion or secretion. We describe here the 1.8 angstrom crystal structure of the universal core of the SRP, revealing protein recognition of a distorted RNA minor groove. Nucleotide analog interference mapping demonstrates the biological importance of observed interactions, and genetic results show that this core is functional in vivo. The structure explains why the conserved residues in the protein and RNA are required for SRP assembly and defines a signal sequence recognition surface composed of both protein and RNA.

• Antson AA
• Single-stranded-RNA binding proteins.
• Curr Opin Struct Biol. 2000; 10: 87-94
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• Our knowledge of protein interactions with RNA molecules has been, so far, largely restricted to cases in which the RNA itself is folded into a secondary and/or tertiary structure stabilised by intramolecular base pairing and stacking. Until recently, only limited structural information has been available about protein interactions with single-stranded RNA. A breakthrough in our understanding of these interactions came in 1999, with the determination of four crystal structures of protein complexes with extended single-stranded RNA molecules. These structures revealed wonderfully satisfying patterns of the ability of proteins to accommodate RNA bases, with the sugar-phosphate backbone often adopting conformations that are different from the classical double helix.

• Wang N, Yamanaka K, Inouye M
• Acquisition of double-stranded DNA-binding ability in a hybrid protein between Escherichia coli CspA and the cold shock domain of human YB-1.
• Mol Microbiol. 2000; 38: 526-34
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• Escherichia coli CspA, a major cold shock protein, is dramatically induced upon temperature downshift. As it binds co-operatively to single-stranded DNA (ssDNA) and RNA without apparent sequence specificity, it has been proposed that CspA acts as an RNA chaperone to facilitate transcription and translation at low temperature. CspA consists of a five-stranded beta-barrel structure containing two RNA-binding motifs, RNP1 and RNP2. Eukaryotic Y-box proteins, such as human YB-1, are a family of nucleic acid-binding proteins that share a region of high homology with CspA (43% identity), termed the cold shock domain (CSD). Their cellular functions are very diverse and are associated with growth-related processes. Here, we replaced the six-residue loop region of CspA between the beta3 and beta4 strands with the corresponding region of the CSD of human YB-1 protein. The resulting hybrid protein became capable of binding to double-stranded DNA (dsDNA) in addition to ssDNA and RNA. The dsDNA-binding ability of an RNP1 point mutant (F20L) of the hybrid was almost unchanged. On the other hand, the dsDNA-binding ability of the hybrid protein was abolished in high salt concentrations in contrast to its ssDNA-binding ability. These results indicate that the loop region between the beta3 and beta4 strands of Y-box proteins, which is a little longer and more basic than that of CspA, plays an important role in their binding to dsDNA.

• Liker E, Fernandez E, Izaurralde E, Conti E
• The structure of the mRNA export factor TAP reveals a cis arrangement of a non-canonical RNP domain and an LRR domain.
• EMBO J. 2000; 19: 5587-98
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• Human TAP is implicated in mRNA nuclear export and is used by simian type D retroviruses to export their unspliced genomic RNA to the cytoplasm of the host cell. We have determined the crystal structure of the minimal TAP fragment that binds the constitutive transport element (CTE) of retroviral RNAs. Unexpectedly, we find the fragment consists of a ribonucleoprotein (RNP) domain, which is not identifiable by its sequence, and a leucine-rich repeat (LRR) domain. The non-canonical RNP domain functions as the general RNA-binding portion of the fragment. The LRR domain is required in cis to the RNP domain for CTE RNA binding. The structural and biochemical properties of the domains point to a remarkable similarity with the U2B"(RNP)-U2A'(LRR) spliceosomal heterodimer. Our in vitro and in vivo functional studies using structure-based mutants suggest that a phylogenetically conserved surface of the LRR domain of TAP may have different roles in the export of viral and cellular RNAs.

• Swairjo MA, Morales AJ, Wang CC, Ortiz AR, Schimmel P
• Crystal structure of trbp111: a structure-specific tRNA-binding protein.
• EMBO J. 2000; 19: 6287-98
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• Trbp111 is a 111 amino acid Aquifex aeolicus structure-specific tRNA-binding protein that has homologous counterparts distributed throughout evolution. A dimer is the functional unit for binding a single tRNA. Here we report the 3D structures of the A.aeolicus protein and its Escherichia coli homolog at resolutions of 2.50 and 1.87 A, respectively. The structure shows a symmetrical dimer of two core domains and a central dimerization domain where the N- and C-terminal regions of Trbp111 form an extensive dimer interface. The core of the monomer is a classical oligonucleotide/oligosaccharide-binding (OB) fold with a five-stranded ss-barrel and a small capping helix. This structure is similar to that seen in the anticodon-binding domain of three class II tRNA synthetases and several other proteins. Mutational analysis identified sites important for interactions with tRNA. These residues line the inner surfaces of two clefts formed between the ss-barrel of each monomer and the dimer interface. The results are consistent with a proposed model for asymmetrical docking of the convex side of tRNA to the dimer.

• Okamura H, Hanaoka S, Nagadoi A, Makino K, Nishimura Y
• Structural comparison of the PhoB and OmpR DNA-binding/transactivation domains and the arrangement of PhoB molecules on the phosphate box.
• J Mol Biol. 2000; 295: 1225-36
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• PhoB is a transcriptional activator that binds to the phosphate box in the promoters of the phosphate genes of Escherichia coli. PhoB contains two functional domains, an N-terminal phosphorylation domain and a C-terminal DNA-binding/transactivation domain. Here, the three-dimensional structure of the DNA-binding/transactivation domain has been determined by NMR. It consists of an N-terminal four-stranded beta-sheet, a central three helical bundle and a C-terminal beta-hairpin. The second and third helices form a helix-turn-helix (HTH) variant containing a longer turn than the corresponding turn of the classical HTH motif. The overall architecture is very close to that of the OmpR DNA-binding/transactivation domain, however, the conformation of the long turn region of PhoB, a putative interaction site for the RNA polymerase sigma subunit, is entirely different from that of the corresponding turn of OmpR, which interacts with the alpha subunit. In addition, the third helix of PhoB is three amino acid residues longer than the corresponding helix of OmpR. The binding site of PhoB is a TGTCA sequence and the phospahte box contains the two binding sites. NMR studies of the complexes of the PhoB DNA-binding/transactivation domain bound to several different DNA molecules have revealed that two PhoB molecules bind in a tandem array on the phosphate box. In each complex of PhoB the third helix of the DNA-binding/transactivation domain is likely to recognize the TGTCA sequence from the major groove of DNA and the C-terminal beta-hairpin contacts on the minor groove of the 3' site out of the TGTCA sequence in a non-specific manner. The long turn region facing outward is likely to interact with the sigma subunit.

• Conte MR et al.
• Structure of tandem RNA recognition motifs from polypyrimidine tract binding protein reveals novel features of the RRM fold.
• EMBO J. 2000; 19: 3132-41
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• Polypyrimidine tract binding protein (PTB), an RNA binding protein containing four RNA recognition motifs (RRMs), is involved in both pre-mRNA splicing and translation initiation directed by picornaviral internal ribosome entry sites. Sequence comparisons previously indicated that PTB is a non-canonical RRM protein. The solution structure of a PTB fragment containing RRMs 3 and 4 shows that the protein consists of two domains connected by a long, flexible linker. The two domains tumble independently in solution, having no fixed relative orientation. In addition to the betaalphabetabetaalphabeta topology, which is characteristic of RRM domains, the C-terminal extension of PTB RRM-3 incorporates an unanticipated fifth beta-strand, which extends the RNA binding surface. The long, disordered polypeptide connecting beta4 and beta5 in RRM-3 is poised above the RNA binding surface and is likely to contribute to RNA recognition. Mutational analyses show that both RRM-3 and RRM-4 contribute to RNA binding specificity and that, despite its unusual sequence, PTB binds RNA in a manner akin to that of other RRM proteins.

• Yakhnin AV, Trimble JJ, Chiaro CR, Babitzke P
• Effects of mutations in the L-tryptophan binding pocket of the Trp RNA-binding attenuation protein of Bacillus subtilis.
• J Biol Chem. 2000; 275: 4519-24
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• The Bacillus subtilis tryptophan biosynthetic genes are regulated by the trp RNA-binding attenuation protein (TRAP). Cooperative binding of L-tryptophan activates TRAP so that it can bind to RNA. The crystal structure revealed that L-tryptophan forms nine hydrogen bonds with various amino acid residues of TRAP. We performed site-directed mutagenesis to determine the importance of several of these hydrogen bonds in TRAP activation. We tested both alanine substitutions as well as substitutions more closely related to the natural amino acid at appropriate positions. Tryptophan binding mutations were identified in vivo having unchanged, reduced, or completely eliminated repression activity. Several of the in vivo defective TRAP mutants exhibited reduced affinity for tryptophan in vitro but did not interfere with RNA binding at saturating tryptophan concentrations. However, a 10-fold decrease in TRAP affinity for tryptophan led to an almost complete loss of regulation, whereas increased TRAP affinity for tryptophan had little or no effect on the in vivo regulatory activity of TRAP. One hydrogen bond was found to be dispensable for TRAP activity, whereas two others appear to be essential for TRAP function. Another mutant protein exhibited tryptophan-independent RNA binding activity. We also found that trp leader RNA increases the affinity of TRAP for tryptophan.

• Lopez MM, Makhatadze GI
• Major cold shock proteins, CspA from Escherichia coli and CspB from Bacillus subtilis, interact differently with single-stranded DNA templates.
• Biochim Biophys Acta. 2000; 1479: 196-202
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• The family of bacterial major cold shock proteins is characterized by a conserved sequence of 65-75 amino acid residues long which form a three-dimensional structure consisting of five beta-sheets arranged into a beta-barrel topology. CspA from Escherichia coli and CspB from Bacillus subtilis are typical representative members of this class of proteins. The exact biological role of these proteins is still unclear; however, they have been implicated to possess ssDNA-binding activity. In this paper, we report the results of a comparative quantitative analysis of ssDNA-binding activity of CspA and CspB. We show that in spite of high homology on the level of primary structure and very similar three-dimensional structures, CspA and CspB have different ssDNA-binding properties. Both proteins preferentially bind polypyrimidine ssDNA templates, but CspB binds to the T-based templates with one order of magnitude higher affinity than to U- or C-based ssDNA, whereas CspA binds T-, U- or C-based ssDNA with comparable affinity. They also show similarities and differences in their binding to ssDNA at high ionic strength. The results of these findings are related to the chemical structure of DNA bases.

• Pasman Z, von Hippel PH
• Regulation of rho-dependent transcription termination by NusG is specific to the Escherichia coli elongation complex.
• Biochemistry. 2000; 39: 5573-85
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• To terminate transcription in E. coli, Rho protein binds an RNA loading site on the nascent transcript, translocates 5'--> 3' along the RNA in an ATP-driven process, and, upon reaching the transcription elongation complex, brings about RNA release. Thus, the Rho-dependent termination process can be viewed, in part, as a kinetic competition between the rate of transcript elongation by RNA polymerase (RNAP) and the rate of Rho translocation along the nascent transcript. In the context of this model, NusG, which is an essential E. coli protein, regulates Rho-dependent termination in an apparently paradoxical way, increasing the rate of transcription elongation of E. coli RNAP in the absence of Rho while also shifting the sites of Rho-dependent termination upstream on the template. Here we investigate the regulation of Rho-dependent termination by NusG. Analytical ultracentrifugation was used to establish the existence of a stable complex of NusG and Rho and to demonstrate a stoichiometry of one NusG monomer per Rho hexamer. Surface plasmon resonance was used to examine the kinetics of the formation and dissociation of the NusG-Rho complex, yielding an association rate constant (k(on)) of 2.8 (+/-0.8) x 10(5) M(-)(1) s(-)(1), a dissociation rate constant (k(off)) of 3.9 (+/-0.7) x 10(-)(3) s(-)(1), and a calculated equilibrium (dissociation) constant (K(d)) of 1.5 (+/-0.3) x 10(-)(8) M. The apparent stability of the NusG-Rho complex is insensitive to changes in salt (potassium acetate) concentration between 0.05 and 0.15 M. The translocation and transcription termination activities of Rho at saturating NusG concentrations were, however, both sensitive to salt concentration over this range, suggesting that these activities do not directly reflect the stability of the NusG-Rho complex. Rho-dependent termination could be demonstrated for transcription complexes in which E. coli RNAP had been substituted by either bacteriophage SP6 or T7 RNAP. NusG, however, was not active in transcription termination assays with either of these phage RNAPs. Thus, we conclude that NusG modulates Rho-dependent termination by interacting specifically with the RNAP of the E. coli elongation complex to render the complex more susceptible to the termination activity of Rho.

• Moy FJ, Glasfeld E, Mosyak L, Powers R
• Solution structure of ZipA, a crucial component of Escherichia coli cell division.
• Biochemistry. 2000; 39: 9146-56
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• ZipA, an essential component of cell division in Escherichia coli, interacts with the FtsZ protein at the midcell in one of the initial steps of septum formation. The high-resolution solution structure of the 144-residue C-terminal domain of E. coli ZipA (ZipA(185)(-)(328)) has been determined by multidimensional heteronuclear NMR. A total of 30 structures were calculated by means of hybrid distance geometry-simulated annealing using a total of 2758 experimental NMR restraints. The atomic root means square distribution about the mean coordinate positions for residues 6-142 for the 30 structures is 0.37 +/- 0.04 A for the backbone atoms, 0. 78 +/- 0.05 A for all atoms, and 0.45 +/- 0.04 A for all atoms excluding disordered side chains. The NMR solution structure of ZipA(185)(-)(328) is composed of three alpha-helices and a beta-sheet consisting of six antiparallel beta-strands where the alpha-helices and the beta-sheet form surfaces directly opposite each other. A C-terminal peptide from FtsZ has been shown to bind ZipA(185)(-)(328) in a hydrophobic channel formed by the beta-sheet providing insight into the ZipA-FtsZ interaction. An unexpected similarity between the ZipA(185)(-)(328) fold and the split beta-alpha-beta fold observed in many RNA binding proteins may further our understanding of the critical ZipA-FtsZ interaction.

• Cupp-Vickery JR, Vickery LE
• Crystal structure of Hsc20, a J-type Co-chaperone from Escherichia coli.
• J Mol Biol. 2000; 304: 835-45
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• Hsc20 is a 20 kDa J-protein that regulates the ATPase activity and peptide-binding specificity of Hsc66, an hsp70-class molecular chaperone. We report herein the crystal structure of Hsc20 from Escherichia coli determined to a resolution of 1.8 A using a combination of single isomorphous replacement (SIR) and multi-wavelength anomalous diffraction (MAD). The overall structure of Hsc20 consists of two distinct domains, an N-terminal J-domain containing residues 1-75 connected by a short loop to a C-terminal domain containing residues 84-171. The structure of the J-domain, involved in interactions with Hsc66, resembles the alpha-topology of J-domain fragments of Escherichia coli DnaJ and human Hdj1 previously determined by solution NMR methods. The C-terminal domain, implicated in binding and targeting proteins to Hsc66, consists of a three-helix bundle in which two helices comprise an anti-parallel coiled-coil. The two domains make contact through an extensive hydrophobic interface ( approximately 650 A(2)) suggesting that their relative orientations are fixed. Thus, Hsc20, in addition to its role in the regulation of the ATPase activity of Hsc66, may also function as a rigid scaffold to facilitate positioning of the protein substrates targeted to Hsc66.

• Keck JL, Roche DD, Lynch AS, Berger JM
• Structure of the RNA polymerase domain of E. coli primase.
• Science. 2000; 287: 2482-6
• Display abstract

• All cellular organisms use specialized RNA polymerases called "primases" to synthesize RNA primers for the initiation of DNA replication. The high-resolution crystal structure of a primase, comprising the catalytic core of the Escherichia coli DnaG protein, was determined. The core structure contains an active-site architecture that is unrelated to other DNA or RNA polymerase palm folds, but is instead related to the "toprim" fold. On the basis of the structure, it is likely that DnaG binds nucleic acid in a groove clustered with invariant residues and that DnaG is positioned within the replisome to accept single-stranded DNA directly from the replicative helicase.

• Frankel AD
• Fitting peptides into the RNA world.
• Curr Opin Struct Biol. 2000; 10: 332-40
• Display abstract

• The structures of several peptide-RNA complexes have been reported in the past year, underscoring the diverse nature of RNA structure and protein interactions. In general, specific peptide conformations are stabilized by the surrounding RNA framework; this is strikingly similar to how peptides are stabilized upon interaction with proteins.

• Yu X, Horiguchi T, Shigesada K, Egelman EH
• Three-dimensional reconstruction of transcription termination factor rho: orientation of the N-terminal domain and visualization of an RNA-binding site.
• J Mol Biol. 2000; 299: 1279-87
• Display abstract

• The Escherichia coli rho transcription termination protein is a hexameric helicase, and is believed to function by separating an RNA-DNA hybrid. Unlike hexameric DNA helicases, where a single strand of DNA passes through the central channel, it has been proposed that the RNA wraps around the outside of the ring. We have generated a three-dimensional reconstruction of rho, and localized a tRNA molecule bound to the primary RNA-binding site to the outside of the ring. An atomic structure of the N-terminal domain of rho fits into our reconstruction uniquely, with the residues involved in RNA-binding on the outside of the ring. Although rho shares a common structural core with the F1-ATPase and other hexameric helicases, there has been a divergence in function due to rho's N-terminal domain, which has no homology to other helicases.

• Altieri AS et al.
• The structure of the transcriptional antiterminator NusB from Escherichia coli.
• Nat Struct Biol. 2000; 7: 470-4
• Display abstract

• We have determined the solution structure of NusB, a transcription antitermination protein from Escherichia coli. The structure reveals a novel, all alpha-helical protein fold. NusB mutations that cause a loss of function (NusB5) or alter specificity for RNA targets (NusB101) are localized to surface residues and likely affect RNA-protein or protein-protein interactions. Residues that are highly conserved among homologs stabilize the protein core. The solution structure of E. coli NusB presented here resembles that of Mycobacterium tuberculosis NusB determined by X-ray diffraction, but differs substantially from a solution structure of E. coli NusB reported earlier.

• Katoh E et al.
• High precision NMR structure of YhhP, a novel Escherichia coli protein implicated in cell division.
• J Mol Biol. 2000; 304: 219-29
• Display abstract

• YhhP, a small protein of 81 amino acid residues encoded by the yhhP gene in the Escherichia coli database, is implicated in cell division although the precise biological function of this protein has not been yet identified. A variety of microorganisms have similar proteins, all of which contain a common CPxP sequence motif in the N-terminal region. We have determined the three-dimensional solution structure of YhhP by NMR spectroscopy in order to obtain insight into its biological function. It folds into a two-layered alpha/beta-sandwich structure with a betaalphabetaalphabetabeta fold, comprising a mixed four-stranded beta-sheet stacked against two alpha-helices, both of which are nearly parallel to the strands of the beta-sheet. The CPxP motif plays a significant structural role in stabilizing the first helix as a part of the new type N-capping box where the Cys-Pro peptide bond adopts a cis configuration. The structure of YhhP displays a striking resemblance to the C-terminal ribosome-binding domain of translation initiation factor IF3 (IF3C). In addition, the surface charge distribution of the RNA-recognition helix of IF3C is nearly the same as that of the corresponding helix of YhhP. These results suggest a structure-based hypothesis in which binding to an RNA target plays an essential role in the function of this ubiquitous protein.

• Mathews II, Kappock TJ, Stubbe J, Ealick SE
• Crystal structure of Escherichia coli PurE, an unusual mutase in the purine biosynthetic pathway.
• Structure Fold Des. 1999; 7: 1395-406
• Display abstract

• BACKGROUND: Conversion of 5-aminoimidazole ribonucleotide (AIR) to 4-carboxyaminoimidazole ribonucleotide (CAIR) in Escherichia coli requires two proteins - PurK and PurE. PurE has recently been shown to be a mutase that catalyzes the unusual rearrangement of N(5)-carboxyaminoimidazole ribonucleotide (N(5)-CAIR), the PurK reaction product, to CAIR. PurEs from higher eukaryotes are homologous to E. coli PurE, but use AIR and CO(2) as substrates to produce CAIR directly. RESULTS: The 1.50 A crystal structure of PurE reveals an octameric structure with 422 symmetry. A central three-layer (alphabetaalpha) sandwich domain and a kinked C-terminal helix form the folded structure of the monomeric unit. The structure reveals a cleft at the interface of two subunits and near the C-terminal helix of a third subunit. Co-crystallization experiments with CAIR confirm this to be the mononucleotide-binding site. The nucleotide is bound predominantly to one subunit, with conserved residues from a second subunit making up one wall of the cleft. CONCLUSIONS: The crystal structure of PurE reveals a unique quaternary structure that confirms the octameric nature of the enzyme. An analysis of the native crystal structure, in conjunction with sequence alignments and studies of co-crystals of PurE with CAIR, reveals the location of the active site. The environment of the active site and the analysis of conserved residues between the two classes of PurEs suggests a model for the differences in their substrate specificities and the relationship between their mechanisms.

• Hermolin J, Dmitriev OY, Zhang Y, Fillingame RH
• Defining the domain of binding of F1 subunit epsilon with the polar loop of F0 subunit c in the Escherichia coli ATP synthase.
• J Biol Chem. 1999; 274: 17011-6
• Display abstract

• We have previously shown that the E31C-substituted epsilon subunit of F1 can be cross-linked by disulfide bond formation to the Q42C-substituted c subunit of F0 in the Escherichia coli F1F0-ATP synthase complex (Zhang, Y., and Fillingame, R. H. (1995) J. Biol. Chem. 270, 24609-24614). The interactions of subunits epsilon and c are thought to be central to the coupling of H+ transport through F0 to ATP synthesis in F1. To further define the domains of interaction, we have introduced additional Cys into subunit epsilon and subunit c and tested for cross-link formation following sulfhydryl oxidation. The results show that Cys, in a continuous stretch of residues 26-33 in subunit epsilon, can be cross-linked to Cys at positions 40, 42, and 44 in the polar loop region of subunit c. The results are interpreted, and the subunit interaction is modeled using the NMR and x-ray diffraction structures of the monomeric subunits together with information on the packing arrangement of subunit c in a ring of 12 subunits. In the model, residues 26-33 form a turn of antiparallel beta-sheet which packs between the polar loop regions of adjacent subunit c at the cytoplasmic surface of the c12 oligomer.

• Eiler S, Dock-Bregeon A, Moulinier L, Thierry JC, Moras D
• Synthesis of aspartyl-tRNA(Asp) in Escherichia coli--a snapshot of the second step.
• EMBO J. 1999; 18: 6532-41
• Display abstract

• The 2.4 A crystal structure of the Escherichia coli aspartyl-tRNA synthetase (AspRS)-tRNA(Asp)-aspartyl-adenylate complex shows the two substrates poised for the transfer of the aspartic acid moiety from the adenylate to the 3'-hydroxyl of the terminal adenosine of the tRNA. A general molecular mechanism is proposed for the second step of the aspartylation reaction that accounts for the observed conformational changes, notably in the active site pocket. The stabilization of the transition state is mediated essentially by two amino acids: the class II invariant arginine of motif 2 and the eubacterial-specific Gln231, which in eukaryotes and archaea is replaced by a structurally non-homologous serine. Two archetypal RNA-protein modes of interactions are observed: the anticodon stem-loop, including the wobble base Q, binds to the N-terminal beta-barrel domain through direct protein-RNA interactions, while the binding of the acceptor stem involves both direct and water-mediated hydrogen bonds in an original recognition scheme.

• Yamanaka K
• Cold shock response in Escherichia coli.
• J Mol Microbiol Biotechnol. 1999; 1: 193-202
• Display abstract

• Sensing a sudden change of the growth temperature, all living organisms produce heat shock proteins or cold shock proteins to adapt to a given temperature. In a heat shock response, the heat shock sigma factor plays a major role in the induction of heat shock proteins including molecular chaperones and proteases, which are well-conserved from bacteria to human. In contrast, no such a sigma factor has been identified for the cold shock response. Instead, RNAs and RNA-binding proteins play a major role in cold shock response. This review describes what happens in the cell upon cold shock, how E. coli responds to cold shock, how the expression of cold shock proteins is regulated, and what their functions are.

• Schindler T, Graumann PL, Perl D, Ma S, Schmid FX, Marahiel MA
• The family of cold shock proteins of Bacillus subtilis. Stability and dynamics in vitro and in vivo.
• J Biol Chem. 1999; 274: 3407-13
• Display abstract

• Bacillus subtilis possesses three homologous small cold shock proteins (CSPs; CspB, CspC, CspD, sequence identity >72%). They share a similar beta-sheet structure, as shown by circular dichroism, and have a very low conformational stability, with CspC being the least stable. Similar to CspB, CspC and CspD unfold and refold extremely fast in a N <==> U two-state reaction with average lifetimes of only 100-150 ms for the native state and 1-6 ms for the unfolded states at 25 degreesC. As a consequence of their low stability and low kinetic protection against unfolding, all three cold shock proteins are rapidly degraded by proteases in vitro. Analysis of the CSP stabilities in vivo by pulse-chase experiments revealed that CspB and CspD are stable during logarithmic growth at 37 degreesC as well as after cold shock. The cellular half-life of CspC is shortened at 37 degreesC, but under cold shock conditions CspC becomes stable. The proteolytic susceptibility of the CSPs in vitro was strongly reduced in the presence of a nucleic acid ligand, suggesting that the observed stabilization of CSPs in vivo is mediated by binding to their substrate mRNA at 37 degreesC and, in particular, under cold shock conditions.

• Lewis HA et al.
• Crystal structures of Nova-1 and Nova-2 K-homology RNA-binding domains.
• Structure Fold Des. 1999; 7: 191-203
• Display abstract

• BACKGROUND: Nova-1 and Nova-2 are related neuronal proteins that were initially cloned using antisera obtained from patients with the autoimmune neurological disease paraneoplastic opsoclonus-myoclonus ataxia (POMA). Both of these disease gene products contain three RNA-binding motifs known as K-homology or KH domains, and their RNA ligands have been identified via binding-site selection experiments. The KH motif structure has been determined previously using NMR spectroscopy, but not using X-ray crystallography. Many proteins contain more than one KH domain, yet there is no published structural information regarding the behavior of such multimers. RESULTS: We have obtained the first X-ray crystallographic structures of KH-domain-containing proteins. Structures of the third KH domains (KH3) of Nova-1 and Nova-2 were determined by multiple isomorphous replacement and molecular replacement at 2.6 A and 2.0 A, respectively. These highly similar RNA-binding motifs form a compact protease-resistant domain resembling an open-faced sandwich, consisting of a three-stranded antiparallel beta sheet topped by three alpha helices. In both Nova crystals, the lattice is composed of symmetric tetramers of KH3 domains that are created by two dimer interfaces. CONCLUSIONS: The crystal structures of both Nova KH3 domains are similar to the previously determined NMR structures. The most significant differences among the KH domains involve changes in the positioning of one or more of the alpha helices with respect to the betasheet, particularly in the NMR structure of the KH1 domain of the Fragile X disease protein FMR-1. Loop regions in the KH domains are clearly visible in the crystal structure, unlike the NMR structures, revealing the conformation of the invariant Gly-X-X-Gly segment that is thought to participate in RNA-binding and of the variable region. The tetrameric arrangements of the Nova KH3 domains provide insights into how KH domains may interact with each other in proteins containing multiple KH motifs.

• Matsuo H, Takagi T
• [mRNA CAP binding protein]
• Tanpakushitsu Kakusan Koso. 1999; 44: 518-29

• Gan E, Richardson JP
• ATP and other nucleotides stabilize the Rho-mRNA complex.
• Biochemistry. 1999; 38: 16882-8
• Display abstract

• Transcription termination factor Rho from Escherichia coli is a protein that consists of a single 47 kDa protomeric unit that can form a hexameric structure. To determine whether active hexamers can form on an RNA by assembly of subunits, we measured the dependence of complex formation on the concentration of Rho protein in the presence and absence of various nucleotides and related the binding properties to association states determined from sedimentation properties. The results show that the presence of adenine nucleotides converts RNA binding from a multimeric process to a largely monomeric process and that the change correlates with the stabilization of multimers of Rho by the nucleotides. The experimental evidence also indicates that the hexameric form of Rho is stabilized slightly by binding to a transcript but that the protein on RNA is in equilibrium with nonhexameric forms. These results suggest that a Rho hexamer can form on a transcript by addition of subunits to a partial assembly, which means that the complex can consist of six subunits surrounding an RNA transcript as proposed in recent models for Rho action.

• Draper DE
• Themes in RNA-protein recognition.
• J Mol Biol. 1999; 293: 255-70
• Display abstract

• Atomic resolution structures are now available for more than 20 complexes of proteins with specific RNAs. This review examines two main themes that appear in this set of structures. A "groove binder" class of proteins places a protein structure (alpha-helix, 310-helix, beta-ribbon, or irregular loop) in the groove of an RNA helix, recognizing both the specific sequence of bases and the shape or dimensions of the groove, which are sometimes distorted from the normal A-form. A second class of proteins uses beta-sheet surfaces to create pockets that examine single-stranded RNA bases. Some of these proteins recognize completely unstructured RNA, and in others RNA secondary structure indirectly promotes binding by constraining bases in an appropriate orientation. Thermodynamic studies have shown that binding specificity is generally a function of several factors, including base-specific hydrogen bonds, non-polar contacts, and mutual accommodation of the protein and RNA-binding surfaces. The recognition strategies and structural frameworks used by RNA binding proteins are not exotically different from those employed by DNA-binding proteins, suggesting that the two kinds of nucleic acid-binding proteins have not evolved independently.

• Krause M, Messer W
• DnaA proteins of Escherichia coli and Bacillus subtilis: coordinate actions with single-stranded DNA-binding protein and interspecies inhibition during open complex formation at the replication origins.
• Gene. 1999; 228: 123-32
• Display abstract

• DnaA-mediated unwinding of the AT-rich region in the replication origins of Escherichia coli and Bacillus subtilis was analysed in vitro with and without single-stranded DNA-binding protein (SSB). In the presence of SSB, the unwound region was larger by a defined number of base pairs. Although the overall structure of the origins is very different, the size and structure of the unwound region were similar. The unwinding reaction at oriC of one organism was inhibited by DnaA protein of the other bacterium. Similarly, hybrid DnaA proteins with swapped DNA-binding domains were inactive and inhibitory to 'open complex' formation at both origins. We suggest that the inhibition is due to inactive mixed complexes.

• Boss A, Nussbaum-Shochat A, Amster-Choder O
• Characterization of the dimerization domain in BglG, an RNA-binding transcriptional antiterminator from Escherichia coli.
• J Bacteriol. 1999; 181: 1755-66
• Display abstract

• The Escherichia coli transcriptional antiterminator protein BglG inhibits transcription termination of the bgl operon in response to the presence of beta-glucosides in the growth medium. BglG is an RNA-binding protein that recognizes a specific sequence partially overlapping the two terminators within the bgl transcript. The activity of BglG is determined by its dimeric state which is modulated by reversible phosphorylation. Thus, only the nonphosphorylated dimer binds to the RNA target site and allows readthrough of transcription. Genetic systems which test dimerization and antitermination in vivo were used to map and delimit the region which mediates BglG dimerization. We show that the last 104 residues of BglG are required for dimerization. Any attempt to shorten this region from the ends or to introduce internal deletions abolished the dimerization capacity of this region. A putative leucine zipper motif is located at the N terminus of this region. The role of the canonical leucines in dimerization was demonstrated by their substitution. Our results also suggest that the carboxy-terminal 70 residues, which follow the leucine zipper, contain another dimerization domain which does not resemble any known dimerization motif. Each of these two regions is necessary but not sufficient for dimerization. The BglG phosphorylation site, His208, resides at the junction of the two putative dimerization domains. Possible mechanisms by which the phosphorylation of BglG controls its dimerization and thus its activity are discussed.

• Correll CC, Wool IG, Munishkin A
• The two faces of the Escherichia coli 23 S rRNA sarcin/ricin domain: the structure at 1.11 A resolution.
• J Mol Biol. 1999; 292: 275-87
• Display abstract

• The sarcin/ricin domain of 23 S - 28 S ribosomal RNA is essential for protein synthesis because it forms a critical part of the binding site for elongation factors. A crystal structure of an RNA of 27 nucleotides that mimics the domain in Escherichia coli 23 S rRNA was determined at 1.11 A resolution. The domain folds into a hairpin distorted by four non-canonical base-pairs and one base triple. The fold is stabilized by cross-strand and intra-stand stacking; no intramolecular stabilizing metal ions are observed. This is the first structure to reveal in great detail the geometry and the hydration of two common motifs that are conserved in this rRNA domain, a GAGA tetraloop and a G-bulged cross-strand A stack. Differences in the region connecting these motifs to the stem in the E. coli and in the rat sarcin/ricin domains may contribute to the species-specific binding of elongation factors. Correlation of nucleotide protection data with the structure indicates that the domain has two surfaces. One surface is accessible, lies primarily in the major groove, and is likely to bind the elongation factors. The second lies primarily in the minor groove, and is likely to be buried in the ribosome. This minor groove surface includes the Watson-Crick faces of the cytosine bases in the unusual A2654.C2666 and U2653.C2667 water-mediated base-pairs.

• Ingham CJ
• Characterisation of the enzymatic and RNA-binding properties of the Rhodobacter sphaeroides 2.4.1. Rho homologue.
• Biochim Biophys Acta. 1999; 1446: 115-25
• Display abstract

• The Escherichia coli Rho is a transcription termination factor with complex enzymatic properties. Rho is a near-universal prokaryotic transcription factor, but very few non-enteric Rho factors have been studied. The expression and enzymatic activity of Rho from the GC-rich, Gram-negative bacterium Rhodobacter sphaeroides was characterised. Poly(C)-activated ATP hydrolysis, multimerisation and the abundance of the R. sphaeroides Rho were similar to the E. coli Rho. The R. sphaeroides Rho was a DNA:RNA helicase. The R. sphaeroides Rho was unique in Rho factors characterised to date in that it did not interact with the lambdatR1 terminator transcript and ATP hydrolysis was unusually weakly activated by poly(U) RNA. A chimeric Rho (RhoER), with the RNA-binding domain from the E. coli Rho and the ATPase domain of the R. sphaeroides Rho, was activated by RNA co-factors in a similar fashion to the E. coli Rho. The activity of RhoER suggests functional interactions between the N- and C-terminal domains of Rho monomers are highly conserved between Rho factors. The main differences between Rho factors from different bacteria is in the specificity of RNA binding although this does not appear to be necessarily dependent on the GC bias of target RNA as has been previously suggested.

• Nagata T et al.
• Structure, backbone dynamics and interactions with RNA of the C-terminal RNA-binding domain of a mouse neural RNA-binding protein, Musashi1.
• J Mol Biol. 1999; 287: 315-30
• Display abstract

• Musashi1 is an RNA-binding protein abundantly expressed in the developing mouse central nervous system. Its restricted expression in neural precursor cells suggests that it is involved in the regulation of asymmetric cell division. Musashi1 contains two ribonucleoprotein (RNP)-type RNA-binding domains (RBDs), RBD1 and RBD2. Our previous studies showed that RBD1 alone binds to RNA, while the binding of RBD2 is not detected under the same conditions. Joining of RBD2 to RBD1, however, increases the affinity to greater than that of RBD1 alone, indicating that RBD2 contributes to RNA-binding. We have determined the three-dimensional solution structure of the C-terminal RBD (RBD2) of Musashi1 by NMR. It folds into a compact alpha beta structure comprising a four-stranded antiparallel beta-sheet packed against two alpha-helices, which is characteristic of RNP-type RBDs. Special structural features of RBD2 include a beta-bulge in beta2 and a shallow twist of the beta-sheet. The smaller 1H-15N nuclear Overhauser enhancement values for the residues of loop 3 between beta2 and beta3 suggest that this loop is flexible in the time-scale of nano- to picosecond order. The smaller 15N T2 values for the residues around the border between alpha2 and the following loop (loop 5) suggest this region undergoes conformational exchange in the milli- to microsecond time-scale. Chemical shift perturbation analysis indicated that RBD2 binds to an RNA oligomer obtained by in vitro selection under the conditions for NMR measurements, and thus the nature of the weak RNA-binding of RBD2 was successfully characterized by NMR, which is otherwise difficult to assess. Mainly the residues of the surface composed of the four-stranded beta-sheet, loops and C-terminal region are involved in the interaction. The appearance of side-chain NH proton resonances of arginine residues of loop 3 and imino proton resonances of RNA bases upon complex formation suggests the formation of intermolecular hydrogen bonds. The structural arrangement of the rings of the conserved aromatic residues of beta2 and beta3 is suitable for stacking interaction with RNA bases, known to be one of the major protein-RNA interactions, but a survey of the perturbation data suggested that the stacking interaction is not ideally achieved in the complex, which may be related to the weaker RNA-binding of RBD2.

• Hermann T, Westhof E
• Non-Watson-Crick base pairs in RNA-protein recognition.
• Chem Biol. 1999; 6: 33543-33543
• Display abstract

• The cellular functions of most RNA molecules involve protein binding, and non-Watson-Crick base pairs are hallmark sites for interactions with proteins. The determination of three-dimensional structures of RNA-peptide and RNA-protein complexes reveals the molecular basis of non-Watson-Crick base-pair recognition.

• Declerck N, Vincent F, Hoh F, Aymerich S, van Tilbeurgh H
• RNA recognition by transcriptional antiterminators of the BglG/SacY family: functional and structural comparison of the CAT domain from SacY and LicT.
• J Mol Biol. 1999; 294: 389-402
• Display abstract

• Transcriptional antiterminators of the BglG/SacY family are regulatory proteins that mediate the induction of sugar metabolizing operons in Gram-positive and Gram-negative bacteria. Upon activation, these proteins bind to specific targets in nascent mRNAs, thereby preventing abortive dissociation of the RNA polymerase from the DNA template. We have previously characterized the RNA-binding domain of SacY from Bacillus subtilis and determined its three-dimensional structure by both NMR and crystallography. In the present study, we have characterized the paralogous domain from LicT and we present the first structural comparison between two BglG/SacY family members. Similar to SacY, the RNA-binding activity of LicT is contained within the 56 N-terminal amino acid residue fragment corresponding to the so-called co-antiterminator (CAT) domain. Surface plasmon resonance affinity measurements show that, compared to SacY-CAT, LicT-CAT binds more tightly and more specifically to its cognate RNA target, with a KD value of about 10(-8) M. The crystal structure of LicT-CAT has been determined at 1.8 A resolution and compared to that of SacY-CAT. Both molecules fold as symmetrical dimers, each monomer comprising a four-stranded antiparallel beta-sheet that stacks against the beta-sheet of the other monomer in a very conserved manner. Comparison of the proposed RNA-binding surfaces shows that many of the conserved atoms concentrate in a central region across one face of the CAT dimer, whereas variable elements are mostly found at the edges. Interestingly, the electrostatic potential maps calculated for the two molecules are quite different, except for the core of the RNA-binding site, which appears essentially neutral in both structures.

• Kim DE, Patel SS
• The mechanism of ATP hydrolysis at the noncatalytic sites of the transcription termination factor Rho.
• J Biol Chem. 1999; 274: 32667-71
• Display abstract

• Escherichia coli transcription termination factor rho is a hexamer with three catalytic subunits that turnover ATP at a fast rate and three noncatalytic subunits that turnover ATP at a relatively slow rate. The mechanism of the ATPase reaction at the noncatalytic sites was determined and was compared with the ATPase mechanism at the catalytic sites. A sequential mechanism for ATP binding or hydrolysis that was proposed for the catalytic sites was not observed at the noncatalytic sites. Pre-steady-state pulse-chase experiments showed that three ATPs were tightly bound to the noncatalytic sites and these were simultaneously hydrolyzed at a rate of 1.8 s(-1) at 18 degrees C. The apparent bimolecular rate constant for ATP binding was determined as 5.4 x 10(5) M(-1) s(-1) in the presence of poly(C) RNA. The ATP hydrolysis products dissociated from the noncatalytic sites at 0.02 s(-1). The hydrolysis of ATP at the noncatalytic sites was at least 130 times slower, and the overall ATPase turnover was 1500 times slower than that at the catalytic sites. These results from studies of the rho protein are likely to be general to hexameric helicases. We propose that the ATPase activity at the noncatalytic site is too slow to drive translocation of the protein on the nucleic acid or to provide energy for nucleic acid unwinding.

• Kurinov IV, Rajamohan F, Venkatachalam TK, Uckun FM
• X-ray crystallographic analysis of the structural basis for the interaction of pokeweed antiviral protein with guanine residues of ribosomal RNA.
• Protein Sci. 1999; 8: 2399-405
• Display abstract

• Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein (RIP), which enzymatically removes a single adenine base from a conserved, surface exposed loop sequence of ribosomal rRNA. We now present unprecedented experimental evidence that PAP can release not only adenine but guanine as well from Escherichia coli rRNA, albeit at a rate 20 times slower than for adenine. We also report X-ray structure analysis and supporting modeling studies for the interactions of PAP with guanine. Our modeling studies indicated that PAP can accommodate a guanine base in the active site pocket without large conformational changes. This prediction was experimentally confirmed, since a guanine base was visible in the active site pocket of the crystal structure of the PAP-guanine complex.

• Ceruso MA, Grottesi A, Di Nola A
• Effects of core-packing on the structure, function, and mechanics of a four-helix-bundle protein ROP.
• Proteins. 1999; 36: 436-46
• Display abstract

• The effects of core-packing on the structure, function and mechanics of the RNA-binding 4-helix-bundle Rop have been studied by molecular dynamics simulations. The structural, dynamical and geometrical properties of the Rop homodimer, (formed by the antiparallel juxtaposition of two helix-turn-helix motifs), have been compared with those of three protein variants described by Munson et al. (Protein Sci, 5:1584-1593, 1996), where the core of the native protein has been systematically repacked using a two-amino acid alphabet: Ala(2)Leu(2)-8, Ala(2)Leu(2)-8-rev, and Leu(2)Ala(2)-8. The results showed that it was possible to readily distinguish the inactive protein Leu(2)Ala(2)-8 from the other functionally active systems based on tertiary and quaternary structure criteria. Structural properties such as native secondary structure content did not correlate with biological activity. Biological activity was related in part to the relative arrangement of the residues within the binding site. But, more global aspects, related to the overall topology of the helical bundle, accounted for the small functional differences between Ala(2)Leu(2)-8 and Ala(2)Leu(2)-8-rev. Mechanically, the 4-helix-bundle absorbed core mutations by altering the local structure at the sequence termini and in the turns that join the two helices of each monomer, and by changing the overall orientation and separation of the extremely rigid helices. Proteins 1999;36:436-446.

• Kim DE, Shigesada K, Patel SS
• Transcription termination factor Rho contains three noncatalytic nucleotide binding sites.
• J Biol Chem. 1999; 274: 11623-8
• Display abstract

• The active form of transcription termination factor rho from Escherichia coli is a homohexamer, but several studies suggest that the six subunits of the hexamer are not functionally identical. Rho has three tight and three weak ATP binding sites. Based on our findings, we propose that the tight nucleotide binding sites are noncatalytic and the weak sites are catalytic. In the presence of RNA, the rho-catalyzed ATPase rate is fast, close to 30 s-1. However, under these conditions the three tightly bound nucleotides dissociate from the rho hexamer at a slow rate of 0.02 s-1, indicating that the three tight nucleotide binding sites of rho do not participate in the fast ATPase turnover. These slowly exchanging nucleotide binding sites of rho are capable of hydrolyzing ATP, but the resulting products (ADP and Pi) bind tightly and dissociate from rho about 1500 times slower than the fast ATPase turnover. Both RNA and excess ATP in solution are necessary for stabilizing nucleotide binding at these sites. In the absence of RNA, or when solution ATP is hydrolyzed to ADP, a faster dissociation of nucleotides was observed. Based on these results, we propose that the rho hexamer is similar to the F1-ATPase and T7 DNA helicase-containing noncatalytic sites that do not participate in the fast ATPase turnover. We propose that the three tight sites on rho are the noncatalytic sites and the three weak sites are the catalytic sites.

• Lopez MM, Yutani K, Makhatadze GI
• Interactions of the major cold shock protein of Bacillus subtilis CspB with single-stranded DNA templates of different base composition.
• J Biol Chem. 1999; 274: 33601-8
• Display abstract

• CspB is a small acidic protein of Bacillus subtilis, the induction of which is increased dramatically in response to cold shock. Although the exact functional role of CspB is unknown, it has been demonstrated that this protein binds single-stranded deoxynucleic acids (ssDNA). We addressed the question of the effect of base composition on the CspB binding to ssDNA by analyzing the thermodynamics of CspB interactions with model oligodeoxynucleotides. Combinations of four different techniques, fluorescence spectroscopy, gel shift mobility assays, isothermal titration calorimetry, and analytical ultracentrifugation, allowed us to show that: 1) CspB can preferentially bind poly-pyrimidine but not poly-purine ssDNA templates; 2) binding to T-based ssDNA template occurs with high affinity (K(d(25 degrees C)) approximately 42 nM) and is salt-independent, whereas binding of CspB to C-based ssDNA template is strongly salt-dependent (no binding is observed at 1 M NaCl), indicating large electrostatic component involved in the interactions; 3) upon binding each CspB covers a stretch of 6-7 thymine bases on T-based ssDNA; and 4) the binding of CspB to T-based ssDNA template is enthalpically driven, indicating the possible involvement of interactions between aromatic side chains on the protein with the thymine bases. The significance of these results with respect to the functional role of CspB in the bacterial cold shock response is discussed.

• Nakamura K, Yahagi S, Yamazaki T, Yamane K
• Bacillus subtilis histone-like protein, HBsu, is an integral component of a SRP-like particle that can bind the Alu domain of small cytoplasmic RNA.
• J Biol Chem. 1999; 274: 13569-76
• Display abstract

• Small cytoplasmic RNA (scRNA) is metabolically stable and abundant in Bacillus subtilis cells. Consisting of 271 nucleotides, it is structurally homologous to mammalian signal recognition particle RNA. In contrast to 4.5 S RNA of Escherichia coli, B. subtilis scRNA contains an Alu domain in addition to the evolutionarily conserved S domain. In this study, we show that a 10-kDa protein in B. subtilis cell extracts has scRNA binding activity at the Alu domain. The in vitro binding selectivity of the 10-kDa protein shows that it recognizes the higher structure of the Alu domain of scRNA caused by five consecutive complementary sequences in the two loops. Purification and subsequent analyses demonstrated that the 10-kDa protein is HBsu, which was originally identified as a member of the histone-like protein family. By constructing a HBsu-deficient B. subtilis mutant, we showed that HBsu is essential for normal growth. Immunoprecipitating cell lysates using anti-HBsu antibody yielded scRNA. Moreover, the co-precipitation of HBsu with (His)6-tagged Ffh depended on the presence of scRNA, suggesting that HBsu, Ffh, and scRNA make a ternary complex and that scRNA serves as a functional unit for binding. These results demonstrated that HBsu is the third component of a signal recognition particle-like particle in B. subtilis that can bind the Alu domain of scRNA.

• Brandi A, Spurio R, Gualerzi CO, Pon CL
• Massive presence of the Escherichia coli 'major cold-shock protein' CspA under non-stress conditions.
• EMBO J. 1999; 18: 1653-9
• Display abstract

• The most characteristic event of cold-shock activation in Escherichia coli is believed to be the de novo synthesis of CspA. We demonstrate, however, that the cellular concentration of this protein is > or = 50 microM during early exponential growth at 37 degrees C; therefore, its designation as a major cold-shock protein is a misnomer. The cspA mRNA level decreases rapidly with increasing cell density, becoming virtually undetectable by mid-to-late exponential growth phase while the CspA level declines, although always remaining clearly detectable. A burst of cspA expression followed by a renewed decline ensues upon dilution of stationary phase cultures with fresh medium. The extent of cold-shock induction of cspA varies as a function of the growth phase, being inversely proportional to the pre-existing level of CspA which suggests feedback autorepression by this protein. Both transcriptional and post-transcriptional controls regulate cspA expression under non-stress conditions; transcription of cspA mRNA is under the antagonistic control of DNA-binding proteins Fis and H-NS both in vivo and in vitro, while its decreased half-life with increasing cell density contributes to its rapid disappearance. The cspA mRNA instability is due to its 5' untranslated leader and is counteracted in vivo by the cold-shock DeaD box RNA helicase (CsdA).

• Chen X, Court DL, Ji X
• Crystal structure of ERA: a GTPase-dependent cell cycle regulator containing an RNA binding motif.
• Proc Natl Acad Sci U S A. 1999; 96: 8396-401
• Display abstract

• ERA forms a unique family of GTPase. It is widely conserved and essential in bacteria. ERA functions in cell cycle control by coupling cell division with growth rate. ERA homologues also are found in eukaryotes. Here we report the crystal structure of ERA from Escherichia coli. The structure has been determined at 2.4-A resolution. It reveals a two-domain arrangement of the molecule: an N-terminal domain that resembles p21 Ras and a C-terminal domain that is unique. Structure-based topological search of the C domain fails to reveal any meaningful match, although sequence analysis suggests that it contains a KH domain. KH domains are RNA binding motifs that usually occur in tandem repeats and exhibit low sequence similarity except for the well-conserved segment VIGxxGxxIK. We have identified a betaalphaalphabeta fold that contains the VIGxxGxxIK sequence and is shared by the C domain of ERA and the KH domain. We propose that this betaalphaalphabeta fold is the RNA binding motif, the minimum structural requirement for RNA binding. ERA dimerizes in crystal. The dimer formation involves a significantly distorted switch II region, which may shed light on how ERA protein regulates downstream events.

• Hard T
• NMR studies of protein-nucleic acid complexes: structures, solvation, dynamics and coupled protein folding.
• Q Rev Biophys. 1999; 32: 57-98

• Langbein I, Bachem S, Stulke J
• Specific interaction of the RNA-binding domain of the bacillus subtilis transcriptional antiterminator GlcT with its RNA target, RAT.
• J Mol Biol. 1999; 293: 795-805
• Display abstract

• Expression of the Bacillus subtilis ptsGHI operon is controlled by transcriptional antitermination mediated by the antiterminator protein GlcT. The antiterminator is inactivated in the absence of glucose, presumably by phosphorylation. A conditional terminator in the ptsG mRNA leader region has been identified. Mutations in this terminator resulted in constitutive expression of the operon. The terminator is overlapped by an inverted repeat (called ribonucleic-antiterminator, RAT) which is thought to form a stem-loop structure upon binding of the antiterminator protein GlcT. The N-terminal 60 amino acid residues of GlcT are able to bind to the RAT and prevent transcriptional termination in vivo. Sequence-specific interaction between the RNA-binding domain and the RAT was demonstrated by surface plasmon resonance analysis. Mutations affecting the RNA-binding domain were isolated and will be discussed with respect to their consequences for dimerization and RNA binding.

• Chen Xp et al.
• Regulatory features of the trp operon and the crystal structure of the trp RNA-binding attenuation protein from Bacillus stearothermophilus.
• J Mol Biol. 1999; 289: 1003-16
• Display abstract

• Characterization of both the cis and trans -acting regulatory elements indicates that the Bacillus stearothermophilustrp operon is regulated by an attenuation mechanism similar to that which controls the trp operon in Bacillus subtilis. Secondary structure predictions indicate that the leader region of the trp mRNA is capable of folding into terminator and anti- terminator RNA structures. B. stearothermophilus also encodes an RNA-binding protein with 77% sequence identity with the RNA-binding protein (TRAP) that regulates attenuation in B. subtilis. The X-ray structure of this protein has been determined in complex with L-tryptophan at 2.5 A resolution. Like the B. subtilis protein, B. stearothermophilus TRAP has 11 subunits arranged in a ring-like structure. The central cavities in these two structures have different sizes and opposite charge distributions, and packing within the B. stearothermophilus TRAP crystal form does not generate the head-to-head dimers seen in the B. subtilis protein, suggesting that neither of these properties is functionally important. However, the mode of L-tryptophan binding and the proposed RNA binding surfaces are similar, indicating that both proteins are activated by l -tryptophan and bind RNA in essentially the same way. As expected, the TRAP:RNA complex from B. stearothermophilus is significantly more thermostable than that from B. subtilis, with optimal binding occurring at 70 degrees C.

• Mao H, Williamson JR
• Local folding coupled to RNA binding in the yeast ribosomal protein L30.
• J Mol Biol. 1999; 292: 345-59
• Display abstract

• The ribosomal protein L30 from yeast Saccharomyces cerevisiae auto-regulates its own synthesis by binding to a structural element in both its pre-mRNA and its mRNA. The three-dimensional structures of L30 in the free (f L30) and the pre-mRNA bound (b L30) forms have been solved by nuclear magnetic resonance spectroscopy. Both protein structures contain four alternating alpha-helices and four beta-strands segments and adopt an overall topology that is an alphabetaalpha three-layer sandwich, representing a unique fold. Three loops on one end of the alphabetaalpha sandwich have been mapped as the RNA binding site on the basis of structural comparison, chemical shift perturbation and the inter-molecular nuclear Overhauser effects to the RNA. The structural and dynamic comparison of f L30 and b L30 reveals that local dynamics may play an important role in the RNA binding. The fourth helix in b L30 is longer than in f L30, and is stabilized by RNA binding. The exposed hydrophobic surface that is buried upon RNA binding may provide the energy necessary to drive secondary structure formation, and may account for the increased stability of b L30.

• Kaan T, Jurgen B, Schweder T
• Regulation of the expression of the cold shock proteins CspB and CspC in Bacillus subtilis.
• Mol Gen Genet. 1999; 262: 351-4
• Display abstract

• The small acidic proteins CspB and CspC are the major cold shock-induced proteins of Bacillus subtilis. Analysis of mRNA revealed a transient four-fold increase in the transcription level of both genes during cold shock. The cspB and cspC mRNAs are dramatically stabilised after a temperature downshift from 37 degrees C to 15 degrees C. The data in this study support the idea that the expression of CspB and CspC in B. subtilis during cold shock is regulated mainly at the post-transcriptional level, as is also the case with CspA in Escherichia coli.

• Gross M et al.
• Formation of amyloid fibrils by peptides derived from the bacterial cold shock protein CspB.
• Protein Sci. 1999; 8: 1350-7
• Display abstract

• Three peptides covering the sequence regions corresponding to the first two (CspB-1), the first three (CspB-2), and the last two (CspB-3) beta-strands of CspB, the major cold shock protein of Bacillus subtilis, have been synthesized and analyzed for their conformations in solution and for their precipitation behavior. The peptides are nearly insoluble in water, but highly soluble in aqueous solutions containing 50% acetonitrile (pH 4.0). Upon shifts of the solvent condition toward lower or higher acetonitrile concentrations, the peptides all form fibrils resembling those observed in amyloid associated diseases. These fibrils have been identified and characterized by electron microscopy, binding of the dye congo red, and X-ray fiber diffraction. Characterization of the peptides in solution by circular dichroism and NMR spectroscopy shows that the formation of these fibrils does not require specific preformed secondary structure in the solution state species. While the majority of the soluble fraction of each peptide is monomeric and unstructured, different types of structures including alpha-helical, beta-sheet, and random coil conformations are observed under conditions that eventually lead to fibril formation. We conclude that the absence of tertiary contacts under solution conditions where binding interactions between peptide units are still favorable is a crucial requirement for amyloid formation. Thus, fragmentation of a sequence, like partial chemical denaturation or mutation, can enhance the capacity of specific protein sequences to form such fibrils.

• Phadtare S, Alsina J, Inouye M
• Cold-shock response and cold-shock proteins.
• Curr Opin Microbiol. 1999; 2: 175-80
• Display abstract

• Both prokaryotes and eukaryotes exhibit a cold-shock response upon an abrupt temperature downshift. Cold-shock proteins are synthesized to overcome the deleterious effects of cold shock. CspA, the major cold-shock protein of Escherichia coli, has recently been studied with respect to its structure, function and regulation at the level of transcription, translation and mRNA stability. Homologues of CspA are present in a number of bacteria. Widespread distribution, ancient origin, involvement in the protein translational machinery of the cell and the existence of multiple families in many organisms suggest that these proteins are indispensable for survival during cold-shock acclimation and that they are probably also important for growth under optimal conditions.

• Feng W, Tejero R, Zimmerman DE, Inouye M, Montelione GT
• Solution NMR structure and backbone dynamics of the major cold-shock protein (CspA) from Escherichia coli: evidence for conformational dynamics in the single-stranded RNA-binding site.
• Biochemistry. 1998; 37: 10881-96
• Display abstract

• The major cold-shock protein (CspA) from Escherichia coli is a single-stranded nucleic acid-binding protein that is produced in response to cold stress. We have previously reported its overall chain fold as determined by NMR spectroscopy [Newkirk, K., Feng, W., Jiang, W., Tejero, R., Emerson, S. D., Inouye, M., and Montelione, G. T. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 5114-5118]. Here we describe the complete analysis of 1H, 13C, and 15N resonance assignments for CspA, together with a refined solution NMR structure based on 699 conformational constraints and an analysis of backbone dynamics based on 15N relaxation rate measurements. An extensive set of triple-resonance NMR experiments for obtaining the backbone and side chain resonance assignments were carried out on uniformly 13C- and 15N-enriched CspA. Using a subset of these triple-resonance experiments, the computer program AUTOASSIGN provided automatic analysis of sequence-specific backbone N, Calpha, C', HN, Halpha, and side chain Cbeta resonance assignments. The remaining 1H, 13C, and 15N resonance assignments for CspA were then obtained by manual analysis of additional NMR spectra. Dihedral angle constraints and stereospecific methylene Hbeta resonance assignments were determined using a new conformational grid search program, HYPER, and used together with longer-range constraints as input for three-dimensional structure calculations. The resulting solution NMR structure of CspA is a well-defined five-stranded beta-barrel with surface-exposed aromatic groups that form a single-stranded nucleic acid-binding site. Backbone dynamics of CspA have also been characterized by 15N T1, T2, and heteronuclear 15N-1H NOE measurements and analyzed using the extended Lipari-Szabo formalism. These dynamic measurements indicate a molecular rotational correlation time taum of 4.88 +/- 0.04 ns and provide evidence for fast time scale (taue < 500 ps) dynamics in surface loops and motions on the microsecond to millisecond time scale within the proposed nucleic acid-binding epitope.

• Tomchick DR, Turner RJ, Switzer RL, Smith JL
• Adaptation of an enzyme to regulatory function: structure of Bacillus subtilis PyrR, a pyr RNA-binding attenuation protein and uracil phosphoribosyltransferase.
• Structure. 1998; 6: 337-50
• Display abstract

• BACKGROUND: The expression of pyrimidine nucleotide biosynthetic (pyr) genes in Bacillus subtilis is regulated by transcriptional attenuation. The PyrR attenuation protein binds to specific sites in pyr mRNA, allowing the formation of downstream terminator structures. UMP and 5-phosphoribosyl-1-pyrophosphate (PRPP), a nucleotide metabolite, are co-regulators with PyrR. The smallest RNA shown to bind tightly to PyrR is a 28-30 nucleotide stem-loop that contains a purine-rich bulge and a putative-GNRA tetraloop. PyrR is also a uracil phosphoribosyltransferase (UPRTase), although the relationship between enzymatic activity and RNA recognition is unclear, and the UPRTase activity of PyrR is not physiologically significant in B. subtilis. Elucidating the role of PyrR structural motifs in UMP-dependent RNA binding is an important step towards understanding the mechanism of pyr transcriptional attenuation. RESULTS: The 1.6 A crystal structure of B. subtilis PyrR has been determined by multiwavelength anomalous diffraction, using a Sm co-crystal. As expected, the structure of PyrR is homologous to those proteins of the large type I PRTase structural family; it is most similar to hypoxanthine-guanine-xanthine PRTase (HGXPRTase). The PyrR dimer differs from other PRTase dimers, suggesting it may have evolved specifically for RNA binding. A large, basic, surface at the dimer interface is an obvious RNA-binding site and uracil specificity is probably provided by hydrogen bonds from mainchain and sidechain atoms in the hood subdomain. These models of RNA and UMP binding are consistent with biological data. CONCLUSIONS: The B. subtilis protein PyrR has adapted the substrate- and product-binding capacities of a PRTase, probably an HGXPRTase, producing a new regulatory function in which the substrate and product are co-regulators of transcription termination. The structure is consistent with the idea that PyrR regulatory function is independent of catalytic activity, which is likely to be extremely low under physiological conditions.

• Huenges M et al.
• Solution structure of the antitermination protein NusB of Escherichia coli: a novel all-helical fold for an RNA-binding protein.
• EMBO J. 1998; 17: 4092-100
• Display abstract

• The NusB protein of Escherichia coli is involved in the regulation of rRNA biosynthesis by transcriptional antitermination. In cooperation with several other proteins, it binds to a dodecamer motif designated rrn boxA on the nascent rRNA. The antitermination proteins of E.coli are recruited in the replication cycle of bacteriophage lambda, where they play an important role in switching from the lysogenic to the lytic cycle. Multidimensional heteronuclear NMR experiments were performed with recombinant NusB protein labelled with 13C, 15N and 2H. The three-dimensional structure of the protein was solved from 1926 NMR-derived distances and 80 torsion angle restraints. The protein folds into an alpha/alpha-helical topology consisting of six helices; the arginine-rich N-terminus appears to be disordered. Complexation of the protein with an RNA dodecamer equivalent to the rrn boxA site results in chemical shift changes of numerous amide signals. The overall packing of the protein appears to be conserved, but the flexible N-terminus adopts a more rigid structure upon RNA binding, indicating that the N-terminus functions as an arginine-rich RNA-binding motif (ARM).

• Hillier BJ, Rodriguez HM, Gregoret LM
• Coupling protein stability and protein function in Escherichia coli CspA.
• Fold Des. 1998; 3: 87-93
• Display abstract

• BACKGROUND: CspA is a small protein that binds single-stranded RNA and DNA. The binding site of CspA consists of a cluster of aromatic amino acids, which form an unusually large nonpolar patch on the surface of the protein. Because nonpolar residues are generally found in the interiors of proteins, this cluster may have evolved to bind nucleic acids at the expense of protein stability. RESULTS: Three neighboring phenylalanines have been mutated singly and in combination to leucine and to serine. All mutations adversely affect DNA binding. Surprisingly, all mutations, and especially those to serine, are destabilizing. CONCLUSIONS: The aromatic cluster in CspA is required not only for protein function but also for protein stability. This result is pertinent to the design of beta-sheet proteins and single-stranded nucleic acid binding proteins, whose binding mode is proposed to be of aromatic-aromatic intercalation.

• Riba I, Gaskell SJ, Cho H, Widger WR, Kohn H
• Evidence for the location of bicyclomycin binding to the Escherichia coli transcription termination factor Rho.
• J Biol Chem. 1998; 273: 34033-41
• Display abstract

• The commercial antibiotic bicyclomycin (Bcm) has been shown to target the essential transcription termination factor Rho in Escherichia coli. Little is known about the Bcm binding domain in Rho. A recent structure-activity relationship study led us to evaluate the reductive amination probe, 5a-(3-formylanilino)dihydrobicyclomycin (FD-Bcm). Biochemical studies showed that FD-Bcm possessed inhibitory activities comparable to Bcm in Rho-dependent ATPase and transcription termination assays. Incubation of Rho with FD-Bcm, ATP, and poly(C) followed by NaBH4 reduction and dialysis led to an appreciable loss of ATPase activity. Inclusion of Bcm with FD-Bcm in the reductive amination reaction protected Rho, indicating that Bcm and FD-Bcm competed for the same binding site in Rho. Incubation of Rho with FD-Bcm and poly(C) followed by NaBH4 reduction provided a sample with residual ATPase activity (12%). Mass spectrometric analysis indicated the presence of two proteins in an approximate 1.2:1 ratio, whose masses corresponded to wild-type Rho (47,010 Da) and lysine-modified Rho (47,417 Da), respectively. Trypsin digestion of the Rho sample followed by high performance liquid chromatography separation and tandem mass spectrometry analysis identified the site of modification as Lys181 within the combined tryptic fragment, Gly-Leu-Ile-Val-Ala-Pro-Pro-Lys-Ala-Gly-Lys (residues 174-184). Similar analysis of a lesser modified sample (following incubation with inclusion of ATP) showed that addition had again occurred at Lys181. These findings provide the first structural information concerning the site of Bcm binding in Rho.

• Zhu AQ, von Hippel PH
• Rho-dependent termination within the trp t' terminator. I. Effects of rho loading and template sequence.
• Biochemistry. 1998; 37: 11202-14
• Display abstract

• About one-half of the terminators of the Escherichia coli genome require transcription termination factor rho to function. Here we use the very "diffuse" trp t' terminator of E. coli to show that both template sequence and transcript secondary structure are involved in controlling the template positions and efficiencies of rho-dependent termination. Termination begins in the wild-type trp t' terminator sequence approximately 97 bps downstream of the promoter under our standard reaction conditions, and termination efficiencies for individual positions on three related templates have been determined in the form of quantitative patterns of rho-dependent RNA release. Comparison of these patterns shows that the rho-dependent termination efficiency at individual template positions depends primarily on the nucleotide sequence at and near the putative 3' end of the transcript, although these efficiencies can also be influenced by RNA sequence elements located further upstream. The amplitudes of the peaks of the RNA release patterns at specific template positions are controlled primarily by the effectiveness of the binding of the rho hexamer to the "rho loading site" of the transcript. Introduction of a stable element of secondary structure into the nascent RNA within the loading site both shifts the position of initial rho-dependent termination downstream and decreases the amplitudes of the peaks of the RNA release pattern at the corresponding sequences. These results and others are consistent with the view that rho-dependent terminators contain two essential components: (i) an upstream rho loading site on the RNA that is 70-80 nucleotide residues in length, essentially devoid of secondary structure, and which contains sufficient numbers of rC residues to activate the RNA-dependent ATPase of rho; and (ii) a downstream sequence within which termination actually occurs. In this study we use the trp t' terminator to characterize the involvement of each of these sequence components in detail in order to provide the parameters required to define a quantitative mechanistic model for the function of rho in transcript termination.

• Nevskaya N et al.
• Crystal structure of ribosomal protein S8 from Thermus thermophilus reveals a high degree of structural conservation of a specific RNA binding site.
• J Mol Biol. 1998; 279: 233-44
• Display abstract

• S8 is one of the core ribosomal proteins. It binds to 16 S RNA with high affinity and independently of other ribosomal proteins. It also acts as a translational repressor in Escherichia coli by binding to its own mRNA. The structure of Thermus thermophilus S8 has been determined by the method of multiple isomorphous replacement at 2.9 A resolution and refined to a crystallographic R-factor of 16.2% (Rfree 27.5%). The two domains of the structure have an alpha/beta fold and are connected by a long protruding loop. The two molecules in the asymmetric unit of the crystal interact through an extensive hydrophobic core and form a tightly associated dimer, while symmetry-related molecules form a joint beta-sheet of mixed type. This type of protein-protein interaction could be realized within the ribosomal assembly. A comparison of the structures of T. thermophilus and Bacillus stearothermophilus S8 shows that the interdomain loop is eight residues longer in the former and reveals high structural conservation of an extensive region, located in the C-terminal domain. From mutational studies this region was proposed earlier to be involved in specific interaction with RNA. On the basis of these data and on the comparison of the two structures of S8, it is proposed that the three-dimensional structure of specific RNA binding sites in ribosomal proteins is highly conserved among different species.

• Yang F, Gustafson KR, Boyd MR, Wlodawer A
• Crystal structure of Escherichia coli HdeA.
• Nat Struct Biol. 1998; 5: 763-4

• Darst SA, Polyakov A, Richter C, Zhang G
• Insights into Escherichia coli RNA polymerase structure from a combination of x-ray and electron crystallography.
• J Struct Biol. 1998; 124: 115-22
• Display abstract

• Our goal is to understand the mechanism of transcription and its regulation. Determining structures of RNA polymerase and transcription complexes is an essential step. Because of their large size and complexity, determination of these structures will require a combination of electron microscopy, biophysical methods, and biochemical methods to identify functionally and structurally relevant subassemblies and domains and x-ray crystallography to determine high-resolution structures of RNA polymerase components and accessory factors. We recently solved the 2.5-A crystal structure of the Escherichia coli RNA polymerase alpha subunit N-terminal domain, which is the first high-resolution structure of a core component required for RNA polymerase assembly and basal transcription. This structure, combined with a new 19-A resolution structure determined by cryo-electron microscopy of helical crystals of E. coli core RNAP embedded in vitreous ice, leads to a model for the organization of the RNAP subunits.

• Schindler T, Perl D, Graumann P, Sieber V, Marahiel MA, Schmid FX
• Surface-exposed phenylalanines in the RNP1/RNP2 motif stabilize the cold-shock protein CspB from Bacillus subtilis.
• Proteins. 1998; 30: 401-6
• Display abstract

• In the cold-shock protein CspB from Bacillus subtilis three exposed Phe residues (F15, F17, and F27) are essential for its function in binding to single-stranded nucleic acids. Usually, the hydrophobic Phe side chains are buried in folded proteins. We asked here whether the exposition of the essential Phe residues could be a cause for the very low conformational stability of CspB. Urea-induced and heat-induced equilibrium unfolding transitions were measured for three mutants of CspB, where Phe 15, Phe 17, and Phe 27 were individually replaced by alanine. Unexpectedly, all three mutations strongly destabilized CspB. The aromatic side chains of Phe 15, Phe 17, and Phe 27 in the active site are thus important for both binding to nucleic acids and conformational stability. There is no compromise between function and stability in the active site. Model calculations indicate that, although they are partially exposed to solvent, all three Phe residues nevertheless lose accessible surface upon folding, and this should favor the native state. A different result is obtained with the F38A variant. Phe 38 is hyperexposed in native CspB, and its substitution by Ala is in fact stabilizing.

• Doevendans PA, Ruiz-Lozano P, van Bilsen M
• Hunting down nucleic acid binding factors in the cardiovascular system.
• Cardiovasc Res. 1998; 38: 301-15
• Display abstract

• Transcription regulation of genes active in the cardiovascular system is a complex process, involving DNA and RNA binding proteins. Nucleic acid binding proteins bind to the regulatory DNA and interact with other proteins, including RNA polymerase to initiate and control the level of transcription. The RNA binding proteins have a function in spliceosome formation and in stabilising mRNA. In this review the currently available molecular approaches to analyse regulatory DNA in relation to DNA binding proteins are discussed. Similar techniques that have been developed for RNA binding protein studies are included. In addition to an explanation of the various methods, examples are provided from DNA-protein interactions on genes active in the cardiovascular system, together with strategies for identification and characterisation of new nucleic acid binding proteins active in cardiac or vascular cell types.

• Carrano L et al.
• Effects of bicyclomycin on RNA- and ATP-binding activities of transcription termination factor Rho.
• Antimicrob Agents Chemother. 1998; 42: 571-8
• Display abstract

• Bicyclomycin is a commercially important antibiotic that has been shown to be effective against many gram-negative bacteria. Genetic and biochemical evidence indicates that the antibiotic interferes with RNA metabolism in Escherichia coli by inhibiting the activity of transcription termination factor Rho. However, the precise mechanism of inhibition is not completely known. In this study we have used in vitro transcription assays to analyze the effects of bicyclomycin on the termination step of transcription. The Rho-dependent transcription termination region located within the hisG cistron of Salmonella typhimurium has been used as an experimental system. The possible interference of the antibiotic with the various functions of factor Rho, such as RNA binding at the primary site, ATP binding, and hexamer formation, has been investigated by RNA gel mobility shift, photochemical cross-linking, and gel filtration experiments. The results of these studies demonstrate that bicyclomycin does not interfere with the binding of Rho to the loading site on nascent RNA. Binding of the factor to ATP is not impeded, on the contrary, the antibiotic appears to decrease the apparent equilibrium dissociation constant for ATP in photochemical cross-linking experiments. The available evidence suggests that this decrease might be due to an interference with the correct positioning of ATP within the nucleotide-binding pocket leading b an inherent block of ATP hydrolysis. Possibly, as a consequence of this interference, the antibiotic also prevents ATP-dependent stabilization of Rho hexamers.

• Kloks CP, Hoffmann A, Omichinski JG, Vuister GW, Hilbers CW, Grzesiek S
• Resonance assignment and secondary structure of the cold shock domain of the human YB-1 protein.
• J Biomol NMR. 1998; 12: 463-4

• Cho HS et al.
• Crystal structure of RNA helicase from genotype 1b hepatitis C virus. A feasible mechanism of unwinding duplex RNA.
• J Biol Chem. 1998; 273: 15045-52
• Display abstract

• Crystal structure of RNA helicase domain from genotype 1b hepatitis C virus has been determined at 2.3 A resolution by the multiple isomorphous replacement method. The structure consists of three domains that form a Y-shaped molecule. One is a NTPase domain containing two highly conserved NTP binding motifs. Another is an RNA binding domain containing a conserved RNA binding motif. The third is a helical domain that contains no beta-strand. The RNA binding domain of the molecule is distinctively separated from the other two domains forming an interdomain cleft into which single stranded RNA can be modeled. A channel is found between a pair of symmetry-related molecules which exhibit the most extensive crystal packing interactions. A stretch of single stranded RNA can be modeled with electrostatic complementarity into the interdomain cleft and continuously through the channel. These observations suggest that some form of this dimer is likely to be the functional form that unwinds double stranded RNA processively by passing one strand of RNA through the channel and passing the other strand outside of the dimer. A "descending molecular see-saw" model is proposed that is consistent with directionality of unwinding and other physicochemical properties of RNA helicases.

• Kranz JK, Hall KB
• RNA binding mediates the local cooperativity between the beta-sheet and the C-terminal tail of the human U1A RBD1 protein.
• J Mol Biol. 1998; 275: 465-81
• Display abstract

• Pairwise coupling theory is applied here to determine the energetic interactions between two elements of the N-terminal RNA binding domain (RBD) of the human U1A protein. The novel application of the theory to this system incorporates both measurements of protein stability and RNA binding to define thermodynamic cycles. In this first example of the application, two regions of the protein are selected for study: tyrosine 13, one of the conserved aromatic residues on the surface of the beta-sheet, and the C-terminal tail of the RBD. The six initial pairwise coupling free energies derived from this system describe the communication between these positions, both in the free and RNA-bound states of the protein. The results show that in the absence of RNA, these two elements of the protein act independently. However, when RNA is bound, there is indirect coupling between Tyr13 and the tail, mediated through the RNA. Subsequent thermodynamic cycles involving additional perturbations to the C-terminal tail further define the communication between the C terminus and the beta-sheet. This work demonstrates the general applicability of the pairwise coupling theory to protein:nucleic acid interactions, and illustrates the necessity of such analyses to describe the network of energetic interactions that comprise RNA recognition by this RBD.

• Walstrom KM, Dozono JM, von Hippel PH
• Effects of reaction conditions on RNA secondary structure and on the helicase activity of Escherichia coli transcription termination factor Rho.
• J Mol Biol. 1998; 279: 713-26
• Display abstract

• The ATPase and helicase activities of the Escherichia coli transcription termination protein rho have been studied under a variety of reaction conditions that alter its transcription termination activity. These conditions include KCl, KOAc, or KGlu concentrations from 50 to 150 mM and Mg(OAc)2 concentrations from 1 to 5 mM (in the presence of 1 mM ATP). In higher KCl or higher Mg(OAc)2 concentrations we found that the translocation of rho hexamers along RNA was slower and less processive than the same process measured at 50 mM monovalent salt concentrations and 1 mM Mg(OAc)2. The ATPase activity of rho was also decreased under reaction conditions that slowed translocation. RNA melting experiments showed that the decreased ATPase activity of rho and the slower helicase activity at increased KCl or Mg(OAc)2 concentrations are accompanied by a concomitant increase in the secondary structure of the RNA portion of the helicase substate. In contrast, the ATPase activity of rho in the presence of poly(rC), a synthetic RNA that does not form salt-concentration-dependent secondary structure, was shown to be the same in each of the three monovalent salts. Thus, the salts do not directly affect the structure or conformation of the rho protein or the binding of rho to single-stranded RNA. However, the translocation of rho along RNA was more processive in 150 mM KOAc or KGlu than in 150 mM KCl, while the RNA secondary structure was the same in all three monovalent salts. Therefore, the monovalent salt present in the reaction may directly affect rho-RNA interactions when the RNA substrate can form secondary structure. Helicase experiments with an RNA molecule that does not contain a rho loading-site showed that rho translocates less processively along this potential helicase substrate. These results suggest that the helicase activity of rho may be significantly regulated by RNA secondary structure. In addition, one of the mechanisms to concentrate the activity of rho on transcripts containing unstructured rho loading sites may be that rho translocation along such molecules is more processive than it is along more structured RNA molecules in the cell.

• Zhang A, Altuvia S, Tiwari A, Argaman L, Hengge-Aronis R, Storz G
• The OxyS regulatory RNA represses rpoS translation and binds the Hfq (HF-I) protein.
• EMBO J. 1998; 17: 6061-8
• Display abstract

• The OxyS regulatory RNA integrates the adaptive response to hydrogen peroxide with other cellular stress responses and protects against DNA damage. Among the OxyS targets is the rpoS-encoded sigma(s) subunit of RNA polymerase. Sigma(s) is a central regulator of genes induced by osmotic stress, starvation and entry into stationary phase. We examined the mechanism whereby OxyS represses rpoS expression and found that the OxyS RNA inhibits translation of the rpoS message. This repression is dependent on the hfq-encoded RNA-binding protein (also denoted host factor I, HF-I). Co-immunoprecipitation and gel mobility shift experiments revealed that the OxyS RNA binds Hfq, suggesting that OxyS represses rpoS translation by altering Hfq activity.

• Jiang W, Hou Y, Inouye M
• CspA, the major cold-shock protein of Escherichia coli, is an RNA chaperone.
• J Biol Chem. 1997; 272: 196-202
• Display abstract

• CspA, the major cold-shock protein of Escherichia coli, is dramatically induced during the cold-shock response. The amino acid sequence of CspA shows 43% identity to the "cold-shock domain" of the eukaryotic Y-box protein family, which interacts with RNA and DNA to regulate their functions. Here, we demonstrate that CspA binds to RNA as a chaperone. First, CspA cooperatively binds to heat-denatured single-stranded RNA if it is larger than 74 bases, causing a supershift in gel electrophoresis. A minimal concentration of CspA at 2.7 x 10(-5) M is absolutely required for this cooperative binding, which is sufficiently lower than the estimated cellular concentration of CspA (10(-4) M) in cold-shocked cells. No specific RNA sequences for CspA binding were identified, indicating that it has a broad sequence specificity for its binding. When the 142-base 5'-untranslated region of the cspA mRNA was used as a substrate for ribonucleases A and T1, the addition of CspA significantly stimulated RNA hydrolysis by preventing the formation of RNase-resistant bands due to stable secondary structures in the 5'-untranslated region. These results indicate that binding of CspA to RNA destabilizes RNA secondary structures to make them susceptible to ribonucleases. We propose that CspA functions as an RNA chaperone to prevent the formation of secondary structures in RNA molecules at low temperature. Such a function may be crucial for efficient translation of mRNAs at low temperatures and may also have an effect on transcription.

• Graumann P, Marahiel MA
• Effects of heterologous expression of CspB, the major cold shock protein of Bacillus subtillis, on protein synthesis in Escherichia coli.
• Mol Gen Genet. 1997; 253: 745-52
• Display abstract

• The major cold shock protein of Bacillus subtilis, CspB, has been shown to affect the level of several cold-induced proteins in B. subtilis after cold shock. Here we show that the expression of CspB in Escherichia coli at 37 degrees C - conditions where the cold shock proteins CspA and CspB of E. coli are not present - resulted in a marked decrease in cellular growth rate and had a profound influence on the pattern of protein synthesis, as revealed by two-dimensional gel electrophoresis. This involves both decreases and increases in the rates of synthesis of specific proteins. Specifically, CspB induction resulted in enhanced beta-galactosidase activity expressed from a transcriptional hns-lacZ fusion. This increase reflects the induction of hns transcription and H-NS synthesis after cold shock, which has been demonstrated to be dependent on CspA in vitro. In contrast, expression of a mutant form of CspB (CspBF15A) that is unable to bind to ssDNA in vitro had no effect on growth rate, pattern of protein synthesis or beta-galactosidase activity. Our data demonstrate a strong influence of CspB on protein synthesis in E. coli and suggest a similar function for CspA in E. coli to that of CspB in B. subtilis.

• Shamoo Y, Krueger U, Rice LM, Williams KR, Steitz TA
• Crystal structure of the two RNA binding domains of human hnRNP A1 at 1.75 A resolution.
• Nat Struct Biol. 1997; 4: 215-22
• Display abstract

• Heterogeneous ribonucleoprotein A1 (hnRNP A1) is an abundant eukaryotic nuclear RNA binding protein. A1 is involved in the packaging of pre-mRNA into hnRNP particles, transport of poly A+ mRNA from the nucleus to the cytoplasm and may modulate splice site selection. The crystal structure of A1(RBD1,2) reveals two independently-folded RNA binding domains (RBDs) connected by a flexible linker. Both RBDs are structurally homologous to the U1A(RBD1), and have their RNA binding platforms oriented in an anti-parallel fashion. The anti-parallel arrangement of the A1 RNA binding platforms suggests mechanisms for RNA condensation and ways of bringing together distant RNA sequences for RNA metabolism such as splicing or transport.

• Walstrom KM, Dozono JM, Robic S, von Hippel PH
• Kinetics of the RNA-DNA helicase activity of Escherichia coli transcription termination factor rho. 1. Characterization and analysis of the reaction.
• Biochemistry. 1997; 36: 7980-92
• Display abstract

• The kinetics of the ATP-dependent RNA-DNA helicase activity of Escherichia colitranscription termination factor rho have been analyzed. Helicase substrates were assembled using 255 nt and 391 nt RNA sequences from the trp t' RNA transcript of E. coli. These RNA sequences each carry a rho "loading site" at a position near the 5'-end, and a rho-dependent terminator sequence at the 3'-end to which complementary approximately 20 nt DNA oligonucleotides have been annealed. A rapid ( approximately 30 s) pre-steady-state burst of helicase activity (DNA oligomer release), followed by a slow linear phase, is observed in reactions carried out at low salt concentrations (50 mM KCl). Using poly(rC) or poly(dC) as traps for the rho that is released after one round of activity, we have shown that the first (burst) phase of the reaction represents the processive translocation of prebound rho hexamers from the rho loading site to the 3'-end of the RNA molecule. The slow phase of the reaction is complex and represents a combination of many different processes, including the slow release of RNA from rho, the reannealing of complementary DNA oligonucleotides to the RNA substrate, and the recycling of rho hexamers onto additional RNA molecules. Reactions carried out at higher salt concentrations (150 mM KCl) consist of only one phase, since under these conditions rho dissociates more rapidly from the RNA, with an amplitude corresponding to several DNA oligomers removed per rho hexamer. Thus, rho can recycle and function as a catalytic helicase under reaction conditions resembling those found in the cell.

• Walstrom KM, Dozono JM, von Hippel PH
• Kinetics of the RNA-DNA helicase activity of Escherichia coli transcription termination factor rho. 2. Processivity, ATP consumption, and RNA binding.
• Biochemistry. 1997; 36: 7993-8004
• Display abstract

• The RNA-binding and RNA-DNA helicase activities of the Escherichia coli transcription termination factor rho have been investigated using natural RNA molecules that are 255 and 391 nucleotide residues in length and that contain the trp t' rho-dependent termination sequence of E. coli. Helicase substrates were prepared from these RNA molecules by annealing one or more DNA oligomers to complementary sequences located at or near the 3'-ends of the RNA molecules to form defined RNA-DNA hybrid sequences ranging in length from 20 to 100 bp. By comparing the fraction of the RNA molecules bound to rho with the fraction of bound DNA oligomers removed from the RNA during one round of the helicase reaction, we have shown that rho translocates processively at 37 degrees C in buffer containing 50 mM KCl. Helicase reactions and ATPase measurements were performed in parallel in the presence of RNA molecules containing RNA-DNA hybrids of various lengths, and we show that both the rate of translocation of the rho hexamer along the RNA chain and the rate of ATP consumption are similar, whether or not DNA is hybridized to the RNA transcript. By combining measurements of translocation and ATPase rates, we estimate that rho consumes approximately 1-2 ATP molecules in translocating over 1 nucleotide residue of the RNA chain at 37 degrees C in 50 mM KCl. The ATPase activity of rho remains the same after one round of the helicase reaction, indicating that rho appears to hydrolyze ATP at the same rate, whether it is translocating along the RNA, separating RNA-DNA hybrids, or bound at the 3'-end of the RNA substrate. We also show that rho binds cooperatively ( approximately 2-4 rho hexamers per RNA chain) to the RNA substrates under our standard helicase reaction conditions. However, cooperative binding is not essential for helicase activity, since this binding stoichiometry can be reduced to approximately 1.5 rho hexamers per 255-nucleotide residue RNA chain by blocking approximately 100 nt of either end of the rho binding site of the helicase substrate with complementary DNA oligonucleotides, with no change in helicase properties. The implications of these results for models of rho helicase function and for the role of rho in termination are discussed.

• Linderoth NA, Tang G, Calendar R
• In vivo and in vitro evidence for an anti-Rho activity induced by the phage P4 polarity suppressor protein Psu.
• Virology. 1997; 227: 131-41
• Display abstract

• The polarity suppression (Psu) protein of bacteriophage P4 causes suppression of transcriptional polarity in Escherichia coli by overcoming Rho termination factor activity. Two new psu mutants defective in polarity suppression are described. The psu5 mutation deletes codons 95-98 from about the middle of the gene, and the mutant protein is inactive. The psu6 mutation changes Phe169 to Val and encodes a temperature-sensitive protein. Constitutive overexpression of psu+ from a plasmid prevents colony formation, but overexpression of mutant genes (psu5, psu6) does not, suggesting that Psu disturbs essential host function(s). Rho protein synthesis is enhanced several-fold in cells containing wild-type Psu, due to readthrough at the rho attenuator, while the physical stability of Rho is maintained. As a consequence, Psu-producing cells accumulate significantly more Rho than normal cells, reminiscent of termination-defective rho mutants. The polarity suppression activity induced by Psu is demonstrated in vitro by the efficient readthrough of Rho-dependent terminators lambda tR1 and TIS2 during coupled transcription-translation. Purified Rho protein restores termination at TIS2 when added to Psu-containing reactions but NusG does not. The data support the hypothesis that Psu has or elicits an anti-Rho function.

• Sozhamannan S, Stitt BL
• Effects on mRNA degradation by Escherichia coli transcription termination factor Rho and pBR322 copy number control protein Rop.
• J Mol Biol. 1997; 268: 689-703
• Display abstract

• Mutants in Escherichia coli transcription termination factor Rho, termed rho(nusD), were previously isolated based on their ability to block the growth of bacteriophage T4. Here we show that rho(nusD) strains have decreased average half-lives for bulk cellular mRNA. Decreased E. coli message lifetimes could be because of increased ribonuclease activity in the rho mutant cells: if a Rho-dependent terminator precedes a ribonuclease gene, weaker termination in the rho mutants could lead to nuclease overexpression. However, inactivation of ribonuclease genes in rho026 cells did not relieve the defective phage growth. Unexpectedly, expression of the pBR322 Rop protein, a structure-specific, sequence-independent RNA-binding protein, in rho(nusD) cells restored the ability of T4 to grow and prolonged cellular message half-life in both the wild-type and the rho026 mutant. These results suggest that it is the RNA-binding ability of Rho rather than its transcription termination function that is important for the inhibition of bacteriophage growth and the shorter bulk mRNA lifetime. We propose that altered interaction of the mutant Rho with mRNA could make the RNA more susceptible to degradation. The inability of the RNA-binding proteins SrmB and DeaD to reverse the rho mutant phenotype when each is overexpressed implies that the required RNA interactions are specific. The results show novel roles for Rho and Rop in mRNA stability.

• Nowatzke WL, Burns CM, Richardson JP
• Function of the novel subdomain in the RNA binding domain of transcription termination factor Rho from Micrococcus luteus.
• J Biol Chem. 1997; 272: 2207-11
• Display abstract

• Transcription termination factor Rho from Micrococcus luteus, a high G + C Gram-positive bacterium, contains an unusual extra sequence within its RNA binding domain that is rich in Arg, Glu, and Asp residues and deficient in hydrophobic residues. To determine the role of this extra sequence, we compared the biochemical properties of a variant lacking nearly all the extra sequence, des(60-300) Rho, to that of wild-type M. luteus Rho. The two forms had very similar properties except that the des(60-300) Rho was unable to terminate transcription with Escherichia coli RNA polymerase at the promoter proximal sites used by the wild-type Rho on a lambda cro DNA template but could cause termination at more distal sites and did cause termination at proximal sites when ITP replaced GTP in the reaction mixture. The RNA binding properties of the two forms of this Rho with normal and inosine-substituted RNAs were found to correlate fully with their termination properties. These results indicate that the arginine-rich extra sequence is directly involved in the selection of the termination site and support the hypothesis that the sequence is present in M. luteus Rho to facilitate its binding to M. luteus transcripts, which are likely to have a high degree of base-paired secondary structure because of their high proportion of G residues.

• Manival X, Yang Y, Strub MP, Kochoyan M, Steinmetz M, Aymerich S
• From genetic to structural characterization of a new class of RNA-binding domain within the SacY/BglG family of antiterminator proteins.
• EMBO J. 1997; 16: 5019-29
• Display abstract

• SacY is the prototype of a family of regulatory proteins able to prevent transcription termination. It interacts with a 29 nucleotide RNA sequence able to fold into a stem-loop structure and partially overlapping with a terminator sequence located in the 5' leader mRNA region of the gene it controls. We show here that the N-terminal fragment of SacY, SacY(1-55), and the corresponding fragments of other members of the family have antiterminator activities with efficiency and specificity identical to those of the full-length proteins. In vitro, this activity correlates with the specific affinity of SacY(1-55) for its RNA target. UV melting experiments demonstrate that SacY(1-55) binding stabilizes the RNA target structure. The NMR solution structure of SacY(1-55) is very similar to that obtained in the crystal (van Tilbeurgh et al., 1997): the peptide is folded as a symmetrical dimer without any structural homology with other RNA-binding domains yet characterized. According to a preliminary NMR analysis of the SacY(1-55)-RNA complex, the protein dimer is not disrupted upon RNA binding and several residues implicated in RNA recognition are located at the edge of the dimer interface. This suggests a new mode of protein-RNA interaction.

• van Tilbeurgh H, Manival X, Aymerich S, Lhoste JM, Dumas C, Kochoyan M
• Crystal structure of a new RNA-binding domain from the antiterminator protein SacY of Bacillus subtilis.
• EMBO J. 1997; 16: 5030-6
• Display abstract

• SacY belongs to a family of, at present, seven bacterial transcriptional antiterminators. The RNA-binding and antitermination capacity of SacY resides in the 55 amino acids at the N-terminal [SacY(1-55)]. The crystal structure at 2 A resolution shows that SacY(1-55) forms a dimer in the crystal, in accordance with the NMR solution structure. The structure of the monomer is a four-stranded beta-sheet with a simple beta1beta2beta3beta4 topology. One side of the sheet is covered by a long surface loop and the other side forms the dimer interface. The dimer is stabilized by the orthogonal stacking of the two beta-sheets. The crystal structure is in excellent agreement with the NMR solution structure (r.m.s. distance for C alpha coordinates is 1.3 A). The structure of SacY(1-55) reveals a new RNA-binding motif.

• Yang M et al.
• Alanine-scanning mutagenesis of Bacillus subtilis trp RNA-binding attenuation protein (TRAP) reveals residues involved in tryptophan binding and RNA binding.
• J Mol Biol. 1997; 270: 696-710
• Display abstract

• In Bacillus subtilis, expression of the trp genes is negatively regulated by an RNA binding protein called TRAP (trp RNA-binding Attenuation Protein), which is activated to bind RNA by binding l-tryptophan. TRAP contains 11 identical subunits assembled in a symmetric ring. We have used alanine-scanning mutagenesis to analyze the functions of surface amino acid residues of TRAP. The in vivo regulatory activity of each mutant TRAP was analyzed in a B. subtilis reporter strain containing a trpE'-'lacZ fusion. Mutant TRAP proteins with defective in vivo regulatory activities were characterized in vitro by measuring their tryptophan binding and RNA binding activities. Most of the mutant proteins with altered tryptophan binding, either affinity or cooperativity, contained substituted residues located on two loops formed by residues 25 to 33 and residues 49 to 52, as well as on the beta-strand and beta-turn contiguous with these loops. Substitution of three residues (Lys37, Lys56 and Arg58) with alanine resulted in significant decreases in the RNA binding activity of TRAP without altering tryptophan binding. Structural analysis shows that these three residues are directly aligned on the outer edge of TRAP. Further mutagenic analysis of these three residues revealed that only lysine or arginine residues at positions 37 or 58 allow proper TRAP function, whereas at position 56, only lysine is functional. Residue Asn20 is the only other residue in TRAP that is located on the line formed by residues 37, 56 and 58, and virtually any amino acid residue is functional at position 20. We propose that RNA wraps around TRAP by interacting with residues Lys37, Lys56 and Arg58.

• Nowatzke WL, Keller E, Koch G, Richardson JP
• Transcription termination factor Rho is essential for Micrococcus luteus.
• J Bacteriol. 1997; 179: 5238-40
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• The growth of Micrococcus luteus, a soil microorganism that belongs to the high-G+C gram-positive phylogenetic group, is prevented by bicyclomycin, an antibiotic that inhibits the activity of the M. luteus transcription termination factor Rho. A mutant that can grow in 0.3 mM bicyclomycin has a Rho that is insensitive to bicyclomycin and has the single amino acid residue change of Asp474 to Gly. These results indicate that the function of its Rho factor is essential for M. luteus and that growth of a gram-positive organism can be blocked by bicyclomycin.

• Hinck AP et al.
• The RNA binding domain of ribosomal protein L11: three-dimensional structure of the RNA-bound form of the protein and its interaction with 23 S rRNA.
• J Mol Biol. 1997; 274: 101-13
• Display abstract

• The three-dimensional solution structure has been determined by NMR spectroscopy of the 75 residue C-terminal domain of ribosomal protein L11 (L11-C76) in its RNA-bound state. L11-C76 recognizes and binds tightly to a highly conserved 58 nucleotide domain of 23 S ribosomal RNA, whose secondary structure consists of three helical stems and a central junction loop. The NMR data reveal that the conserved structural core of the protein, which consists of a bundle of three alpha-helices and a two-stranded parallel beta-sheet four residues in length, is nearly the same as the solution structure determined for the non-liganded form of the protein. There are however, substantial chemical shift perturbations which accompany RNA binding, the largest of which map onto an extended loop which bridges the C-terminal end of alpha-helix 1 and the first strand of parallel beta-sheet. Substantial shift perturbations are also observed in the N-terminal end of alpha-helix 1, the intervening loop that bridges helices 2 and 3, and alpha-helix 3. The four contact regions identified by the shift perturbation data also displayed protein-RNA NOEs, as identified by isotope-filtered three-dimensional NOE spectroscopy. The shift perturbation and NOE data not only implicate helix 3 as playing an important role in RNA binding, but also indicate that regions flanking helix 3 are involved as well. Loop 1 is of particular interest as it was found to be flexible and disordered for L11-C76 free in solution, but not in the RNA-bound form of the protein, where it appears rigid and adopts a specific conformation as a result of its direct contact to RNA.

• Horiguchi T, Miwa Y, Shigesada K
• The quaternary geometry of transcription termination factor rho: assignment by chemical cross-linking.
• J Mol Biol. 1997; 269: 514-28
• Display abstract

• Transcription termination factor rho from Escherichia coli is a ring-shaped homohexamer of 419 amino acid subunits and catalyzes an ATP-dependent release of nascent RNA transcripts. Previous chemical cross-linking studies suggested that the rho hexamer might have D3 symmetry with three isologous dimers as protomers. However, our recent mutational analysis of rho alongside its putative structural homology to F1-ATPase rather argued for C6 symmetry. To resolve this discrepancy, we have re-investigated the pattern of cross-linking of rho using various cross-linkers with different functional groups and spacer lengths. Upon reaction with dimethyl suberimidate followed by SDS-polyacrylamide gel electrophoresis, rho protein generated a series of cross-linked oligomers up to hexamers, of which dimers migrated as distinct doublet bands of approximately equal intensities. However, the lower band became much stronger than the upper one with dimethyl adipimidate and difluorodinitrobenzene, and vice versa with disuccinimidyl glutarate, disuccinimidyl suberate and disulfosuccinimidyl tartarate. Furthermore, the trimeric products also produced doublet bands, whose relative intensities were again variable with cross-linkers, but in an inverse correlation with those of the dimer bands. These results combined with theoretical considerations support a C6 symmetry model in which cross-linking is assumed to occur stochastically at one of two alternative sites within each subunit interface with variable relative frequencies depending on cross-linkers. The D3 symmetry is excluded, for the putative trimeric subspecies should always retain mutually equal intensities in that case. Detailed inspections of the cross-linking kinetics further revealed a moderate characteristic of C3 symmetry for the rho hexamer such that the collective as well as relative rates of cross-linking at the two available sites could fluctuate between alternating interfaces. The final model designated as C3/6 is also compatible with other functional and structural properties known for rho.

• Krause M, Ruckert B, Lurz R, Messer W
• Complexes at the replication origin of Bacillus subtilis with homologous and heterologous DnaA protein.
• J Mol Biol. 1997; 274: 365-80
• Display abstract

• The initial steps in the formation of the initiation complex at oriC of Bacillus subtilis were analyzed with special emphasis on the exchangeability of B. subtilis DnaA protein by DnaA of Escherichia coli. The DNA binding domain of B. subtilis DnaA protein was localized in the 93 C-terminal amino acids. Formation of the "initial complex", as analyzed by electron microscopy, was indistinguishable with B. subtilis DnaA protein or with E. coli DnaA. Similarly, both proteins were able to form loops by interaction of DnaA proteins bound to the DnaA box regions upstream and downstream of the dnaA gene in B. subtilis oriC. The region of local unwinding in the "open complex" was precisely defined. It is located at one side of a region of helical instability, a DNA unwinding element (DUE). Unwinding in oriC could only be catalyzed by the homologous DnaA protein.

• Manival X, Aymerich S, Strub MP, Dumas C, Kochoyan M, van Tilbeurgh H
• Crystallization of the RNA-binding domain of the transcriptional antiterminator protein SacY from Bacillus subtilis.
• Proteins. 1997; 28: 590-4
• Display abstract

• SacY is the antiterminator protein involved in the induction by sucrose of the expression of the levansucrase gene (sacB) of Bacillus subtilis. In the presence of sucrose, SacY is activated and prevents premature termination of transcription by binding to a RNA-antiterminator (RAT) sequence partially overlapping with the terminator sequence. SacY consists of a RNA-binding N-terminal domain, SacY(1-55), and a regulatory domain, SacY(56-280), sensitive to the sucrose concentration. SacY(1-55) is in itself capable of binding to the RAT sequence and preventing termination independently of the sucrose concentration. In this paper we describe the overexpression, the purification, and the crystallization of SacY(1-55). We obtained six different crystal forms, some of them diffracting to high resolution (> 1.5 A). Self rotation function calculations indicated the presence of a dimer in the asymmetric unit, which is in agreement with a proposed oligomeric state in solution as observed by high-resolution NMR measurements. The crystallization of some site-directed cysteine mutants opens the way of solving the structure by multiple isomorphous replacement.

• Lee CA
• Type III secretion systems: machines to deliver bacterial proteins into eukaryotic cells?
• Trends Microbiol. 1997; 5: 148-56
• Display abstract

• Type III secretion systems in certain bacterial pathogens are induced upon contact with host cells and directly deliver virulence proteins into the host cell cytosol. The increasing number of Gram-negative bacterial pathogens discovered to encode type III secretion systems raises interesting questions. Are type III systems generic machines that deliver virulence proteins into host cells? Is contact with host cells a common regulatory cue for type III systems?

• Bycroft M, Hubbard TJ, Proctor M, Freund SM, Murzin AG
• The solution structure of the S1 RNA binding domain: a member of an ancient nucleic acid-binding fold.
• Cell. 1997; 88: 235-42
• Display abstract

• The S1 domain, originally identified in ribosomal protein S1, is found in a large number of RNA-associated proteins. The structure of the S1 RNA-binding domain from the E. coli polynucleotide phosphorylase has been determined using NMR methods and consists of a five-stranded antiparallel beta barrel. Conserved residues on one face of the barrel and adjacent loops form the putative RNA-binding site. The structure of the S1 domain is very similar to that of cold shock protein, suggesting that they are both derived from an ancient nucleic acid-binding protein. Enhanced sequence searches reveal hitherto unidentified S1 domains in RNase E, RNase II, NusA, EMB-5, and other proteins.

• Kopylov AM
• [X-ray structural analysis of RNA-protein complexes]
• Biokhimiia. 1996; 61: 1911-6

• Mayr B, Kaplan T, Lechner S, Scherer S
• Identification and purification of a family of dimeric major cold shock protein homologs from the psychrotrophic Bacillus cereus WSBC 10201.
• J Bacteriol. 1996; 178: 2916-25
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• Whole-cell protein patterns of a psychrotrophic Bacillus cereus strain from cultures grown at 7 and 30 degrees C were compared. This analysis revealed that at least three major proteins are expressed at a significantly higher rate at 7 degrees C than at 30 degrees C. The most abundant of these cold-induced proteins was a small polypeptide of 7.5 kDa, designated CspA, of B. cereus. In addition, four small proteins very similar in size to CspA were seen on both 7 degrees C and 30 degrees C two-dimensional protein gels. Immunoblot analysis using B. cereus anti-CspA antibodies indicated that the five proteins described above plus an additional sixth protein not visible on silver-stained two-dimensional gels are members of a B. cereus cold shock protein family. This hypothesis was corroborated by cloning and sequencing of the genes encoding five proteins of this family. The protein sequences deduced are highly similar and show homology to small procaryotic cold shock proteins and to the cold shock domain of eucaryotic Y-box proteins. Besides CspA, only one of the additional five CspA homologs was slightly cold inducible. In the presence of 100 mM NaCl, the two purified members of the protein family (CspA and CspE) elute as dimers at an apparent molecular mass of 15 kDa from a gel filtration column. At higher salt concentrations, they dissociate into their monomers. Their ability to bind to the ATTGG motif of single-stranded oligonucleotides was demonstrated by band shift analysis.

• Nowatzke WL, Richardson JP
• Characterization of an unusual Rho factor from the high G + C gram-positive bacterium Micrococcus luteus.
• J Biol Chem. 1996; 271: 742-7
• Display abstract

• A transcription termination factor (Rho) was purified from the Gram-positive bacterium Micrococcus luteus, and the complete gene sequence was determined. The M. luteus Rho polypeptide has 690 residues, which is 271 residues more than its homolog from Escherichia coli. Most of the additional residues compose a highly charged, hydrophilic segment that is inserted in a non-conserved region between two conserved regions of the RNA-binding domain of the known Rho homolog proteins. This segment extends from residues 49 to 311 and includes a stretch of 238 residues that contain no hydrophobic side chains. Biochemical studies indicate that the M. luteus protein is very similar to E. coli Rho in terms of its RNA-dependent NTPase activity and its sensitivity to the Rho-specific inhibitor bicyclomycin. However, the M. luteus protein has a less stringent RNA cofactor specificity. It also acts to terminate RNA transcription with E. coli RNA polymerase on the lambda cro DNA template, but at much earlier termination stop points than those recognized by E. coli Rho. Thus, the M. luteus protein functions as a true Rho factor, but with a different specificity than that of E. coli Rho. We propose that this altered specificity is consistent with its need to function on transcripts that have a high content of G + C residues.

• Stitt BL, Kempner ES
• Structure-function relationships in Escherichia coli transcription termination protein Rho revealed by radiation target analysis.
• Arch Biochem Biophys. 1996; 334: 268-76
• Display abstract

• High-energy electrons were used to measure the target sizes for inactivation of the RNA-dependent ATPase activity of Escherichia coli transcription termination factor Rho, for its ATP binding ability, and for its physical destruction. SDS-PAGE analysis of irradiated samples indicated that the target size for polypeptide destruction in the homohexameric enzyme is the dimer, indicating that energy transfer must occur from a hit subunit to one other subunit, although the subunits are not known to be linked by any covalent bonds. The ATP binding ability of Rho also inactivates as a dimer, a result that is consistent with the physical destruction target size. However, a single subunit as the ATP binding entity is not excluded. The RNA-dependent ATPase activity of Rho inactivates with the apparent target size of trimer to tetramer, indicating that interactions among the subunits of Rho are required for ATP hydrolysis. Rho hexamers are known to exchange subunits, although the identity of the exchanging unit is not known. Models in which this property of Rho is taken into account indicate that the closest fit to the experimental data is for an ATPase target size of a hexamer with dimers as the exchanging units, consistent with earlier chemical inactivation studies.

• Graumann P, Marahiel MA
• A case of convergent evolution of nucleic acid binding modules.
• Bioessays. 1996; 18: 309-15
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• Divergent evolution can explain how many proteins containing structurally similar domains, which perform a variety of related functions, have evolved from a relatively small number of modules or protein domains. However, it cannot explain how protein domains with similar, but distinguishable, functions and similar, but distinguishable, structures have evolved. Examples of this are the RNA-binding protein containing the RNA-binding domain (RBD), and a newly established protein group, the cold-shock domain (CSD) protein family. Both protein domains contain conserved RNP motifs on similar single-stranded nucleic acid-binding surfaces. Apart from the RNP motifs, which have a similar function, the two families show little similarity in topology or amino acid sequence. This can be considered an interesting example of convergent evolution at the molecular level. Previously, a beta-sheet surface was found to interact with RNA in non-homologous proteins from yeast, phage and man, revealing that this mode of RNA binding may be a widely recurring theme.

• Nagai K
• RNA-protein complexes.
• Curr Opin Struct Biol. 1996; 6: 53-61
• Display abstract

• The three commonly found RNA-binding domains, the ribonucleoprotein (RNP) domain, the double stranded RNA binding domain (dsRBD) and the K homology (KH) domain, have now been shown to have an alpha/beta fold similar to that found in many ribosomal proteins. Crystal structures of two hairpin RNA-protein complexes have been determined recently: the U1A spliceosomal protein bound to hairpin II of U1 small nuclear RNA, and MS2 bacteriophage capsid protein bound to a hairpin present at the ribosomal binding site of MS2 replicase mRNA. The crystal structure of the tryptophan operon RNA binding attenuation protein from Bacillus subtilis shows a novel structure with 11 monomers arranged in a doughnut-shaped ring that binds 11 copies of (U/G)AG triplets presented in the leader sequence of the tryptophan operon polycistronic message.

• Jones PG, Inouye M
• RbfA, a 30S ribosomal binding factor, is a cold-shock protein whose absence triggers the cold-shock response.
• Mol Microbiol. 1996; 21: 1207-18
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• The cold-shock response, characterized by a specific pattern of gene expression, is induced upon a downshift in temperature and in the presence of inhibitors of ribosomal function. Here, we demonstrate that RbfA of Escherichia coli, considered to be involved in ribosomal maturation and/or initiation of translation, is a cold-shock protein. Shifting the rbfA mutant to a lower temperature resulted in a constitutive induction of the cold-shock response accompanied by slower growth at low temperatures, while shifting the rbfA mutant that overproduces wild-type RbfA resulted in an increase in total protein synthesis accompanied by faster growth adaptation to the lower temperature. Furthermore, the cold-shock response was also constitutively induced in a cold-sensitive 16S rRNA mutant at low temperatures. Accompanying the transient induction of the cold-shock response, we also report that shifting E. coli from 37 degrees C to 15 degrees C resulted in a temporary inhibition of initiation of translation, as evidenced by the transient decrease in polysomes accompanied by the transient increase in 70S monosomes. The accumulative data indicate that the inducing signal for the cold-unadapted non-translatable ribosomes which are converted to cold-adapted translatable ribosomes by the association of cold-shock proteins such as RbfA. Therefore, the expression of the cold-shock response, and thus cellular adaptation to low temperature, is regulated at the level of translation. The data also indicate that cold-shock proteins can be translated by ribosomes under conditions that are not translatable for most mRNAs.

• Martinez A, Burns CM, Richardson JP
• Residues in the RNP1-like sequence motif of Rho protein are involved in RNA-binding affinity and discrimination.
• J Mol Biol. 1996; 257: 909-18
• Display abstract

• The termination of transcription in Escherichia coli by action of Rho factor is dependent on the ability of this homohexameric protein to make productive interactions with the nascent RNA molecule to be terminated. The roles of two residues in a phylogenetically conserved sequence motif in the RNA-binding domain of Rho, Asp60 and Phe62, were analyzed by studies of the biochemical properties of pure mutant proteins. F62S Rho had greatly reduced affinity for lambda cro RNA, very poor ability to terminate transcription in vitro by itself and only partial termination activity (at a level consistent with its in vivo defect) in the presence of NusG. D60G Rho had a high affinity for lambda cro RNA but a much lower ability to discriminate against RNA molecules lacking cis-acting Rho-utilization sequences, and a reduced efficiency of termination that was not improved by NusG. These results indicate a major role for Phe62 in stabilizing the binding of Rho to RNA through hydrophobic interactions, while Asp60 provides an electrostatic repulsive force that allows a rapid dissociation of non-productive complexes with RNA.

• Martinez A, Opperman T, Richardson JP
• Mutational analysis and secondary structure model of the RNP1-like sequence motif of transcription termination factor Rho.
• J Mol Biol. 1996; 257: 895-908
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• The function of transcription termination factor Rho from Escherichia coli is dependent upon its ability to bind to specific sites on nascent RNA molecules. The roles of 19 individual amino acid residues (Ile49 to Ser67) in and near a phylogenetically conserved sequence segment of Rho that is similar to the RNP1 motif found in many RNA-binding proteins were examined by testing the phenotypic consequences of mutational changes that were introduced into rho by a random-sequence cassette mutagenesis procedure. The tests of each mutant included the ability of the cells to survive at 42 degrees C in the absence of wild-type rho, the efficiency of termination at a Rho-dependent terminator (lambdatR1) in vivo, the relative level of expression of the mutant protein, and the ability of some of the mutant proteins to bind RNA. The results revealed that residues in the RNP1-like sequence of DGFGFLR (residues 60 to 66) were more important than residues 49 to 59 for termination function and RNA binding, and identified three residues that were particularly sensitive to mutation: Asp60, Phe62 and Arg66. The properties of the mutants are consistent with a secondary structure model, derived from phylogenetic analysis, that has the RNP1-like sequence on one of the three beta-strands of an antiparallel beta-sheet with Asp60 and Gly61 in a turn and the side-chains of Phe62, Phe64 and Arg66 accessible on the same face of the beta-structure for interaction with RNA.

• Avis JM, Allain FH, Howe PW, Varani G, Nagai K, Neuhaus D
• Solution structure of the N-terminal RNP domain of U1A protein: the role of C-terminal residues in structure stability and RNA binding.
• J Mol Biol. 1996; 257: 398-411
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• The solution structure of a fragment of the human U1A spliceosomal protein containing residues 2 to 117 (U1A117) determined using multi-dimensional heteronuclear NMR is presented. The C-terminal region of the molecule is considerably more ordered in the free protein than thought previously and its conformation is different from that seen in the crystal structure of the complex with U1 RNA hairpin II. The residues between Asp90 and Lys98 form an alpha-helix that lies across the beta-sheet, with residues IIe93, IIe94 and Met97 making contacts with Leu44, Phe56 and IIe58. This interaction prevents solvent exposure of hydrophobic residues on the surface of the beta-sheet, thereby stabilising the protein. Upon RNA binding, helix C moves away from this position, changing its orientation by 135 degrees to allow Tyr13, Phe56 and Gln54 to stack with bases of the RNA, and also allowing Leu44 to contact the RNA. The new position of helix C in the complex with RNA is stabilised by hydrophobic interactions from IIe93 and IIe94 to IIe58, Leu 41, Val62 and His 10, as well as a hydrogen bond between Ser91 and Thr11. The movement of helix C mainly involves changes in the main-chain torsion angles of Thr89, Asp90 and Ser91, the helix thereby acting as a "lid" over the RNA binding surface.

• Washburn RS, Stitt BL
• In vitro characterization of transcription termination factor Rho from Escherichia coli rho(nusD) mutants.
• J Mol Biol. 1996; 260: 332-46
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• Escherichia coli nusD strains are bacteria that carry mutations in rho, the gene for transcription termination factor Rho, that block the growth of phages T4 and lambdar32. We have identified the rho mutation in six independent nusD strains, and although five of the strains have different mutations, with one exception the mutations are in the proposed RNA-binding domain of Rho. We overexpressed, purified, and characterized the five different mutant Rho proteins. All show substantial RNA-dependent ATPase activity with several homoribopolymers or the lambda cro message as cofactor. At the lambda tR1 Rho-dependent terminator in vitro, all mutant Rho proteins show decreased termination compared with wild-type, and all also terminate within cro at a new terminator, tRE, with endpoints 5' to tR1 at 170, 200, 245 and 260 nucleotides 3' from the transcription start. The mutant Rho proteins are proposed to interfere with bacteriophage T4 growth through indirect effects on host gene expression.

• Graumann P, Marahiel MA
• Some like it cold: response of microorganisms to cold shock.
• Arch Microbiol. 1996; 166: 293-300
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• Bacteria respond to an abrupt decrease in temperature with a specific response, in which cold-induced proteins (CIPs) are transiently expressed at a higher level. Employing two-dimensional gel electrophoresis, several CIPs have been identified. In spite of this, the overall function of the cold shock response is unclear. Recently, the main attention has focused on a group of conserved cold shock proteins (CSPs) that have been shown to have the highest induction after cold shock and to play a major regulatory role in the physiology of adaptation to low temperatures. CSPs, of which Escherichia coli, Bacillus subtilis, and B. cereus possess a family comprising at least 3-7 proteins, are small acidic proteins that share over 45% of sequence identity. Recent evidence suggests that members of this wide-spread protein family can function both at the transcriptional and translational level in vitro. However, the exact mode of action has yet to be established. In addition, post-transcriptional regulation seems to play a major role in the induction of CSPs, a process in which the ribosome may be involved. This is in accordance with a model in which the ribosome has been proposed to be the sensor of temperature in bacteria.

• Richardson JP
• Structural organization of transcription termination factor Rho.
• J Biol Chem. 1996; 271: 1251-4

• Briercheck DM, Allison TJ, Richardson JP, Ellena JF, Wood TC, Rule GS
• 1H, 15N and 13C resonance assignments and secondary structure determination of the RNA-binding domain of E.coli rho protein.
• J Biomol NMR. 1996; 8: 429-44
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• Protein fragments containing the RNA-binding domain of Escherichia coli rho protein have been over-expressed in E. coli. NMR spectra of the fragment containing residues 1-116 of rho protein (Rho116) show that a region of this protein is unfolded in solution. Addition of (dC)10 to this fragment stabilizes the folded form of the protein. The fragment comprising residues 1-130 of rho protein (Rho130) is found to be stably folded, both in absence and presence of nucleic acid. NMR studies of the complex of Rho130 with RNA and DNA oligonucleotides indicate that the binding-site size, affinity, and specificity of Rho130 are similar to those of intact rho protein; therefore, Rho130 is a suitable model of the RNA-binding domain of Rho protein. NMR line widths as well as titration experiments of Rho130 complexed with oligonucleotides of various lengths suggests that Rho130 forms oligomers in the presence of longer oligonucleotides. 1H, 15N and 13C resonance assignments were facilitated by the utilization of two pulse sequences, CN-NOESY and CCH-TOCSY. The secondary structure of unliganded Rho130 has been determined by NMR techniques, and it is clear that the RNA-binding domain of rho is more structurally similar to the cold shock domain than to the RNA recognition motif.

• Magyar A, Zhang X, Kohn H, Widger WR
• The antibiotic bicyclomycin affects the secondary RNA binding site of Escherichia coli transcription termination factor Rho.
• J Biol Chem. 1996; 271: 25369-74
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• The interaction of Rho and the antibiotic bicyclomycin was probed using in vitro transcription termination reactions, poly(C) binding assays, limited tryptic digestions, and the bicyclomycin inhibition kinetics of ATPase activity in the presence of poly(dC) and ribo(C)10. The approximate I50 value for the bicyclomycin inhibition of transcription termination at Rho-dependent sites within a modified trp operon template was 5 microM. At antibiotic concentrations near the I50 value, bicyclomycin inhibition of Rho-dependent transcripts was accompanied by the appearance of a new set of transcripts whose size was midway between the Rho-dependent transcripts and the readthrough transcripts. Bicyclomycin did not inhibit poly(C) binding to Rho. In the presence of poly(dC), bicyclomycin showed a reversible mixed inhibition of the ribo(C)10-stimulated ATPase activity. The extrapolated Ki for bicyclomycin was 2.8 microM without ribo(C)10 and increased to 26 microM in the presence of ribo(C)10. Correspondingly, the Km(app) for ribo(C)10 without bicyclomycin was 0.8 microM and with bicyclomycin was 5 microM at infinite inhibitor concentration. The data suggested that the antibiotic binds to Rho, influencing the secondary RNA binding (tracking) site on Rho and slows the tracking of Rho toward the bound RNA polymerase.

• Etchegaray JP, Jones PG, Inouye M
• Differential thermoregulation of two highly homologous cold-shock genes, cspA and cspB, of Escherichia coli.
• Genes Cells. 1996; 1: 171-8
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• BACKGROUND: The major cold-shock protein in Escherichia coli is CspA, a 7.4 kDa protein. A CspA family has been found which consists of four additional proteins, CspB, CspC, CspD and CspE. The expression of cspB, unlike the other homologues, is cold-shock inducible like cspA. RESULTS: We examined the cold-shock induction of CspA and CspB at various temperatures. The cspA induction is observed by temperature shift from 37 to 30 degrees C and high levels of CspA production are observed between 24 and 10 degrees C. In contrast, CspB production occurs only by temperature shift to below 20 C, with maximum induction at 15 degrees C. Both cspA and cspB expressions were found to be induced at the level of transcription as determined by primer extension. CONCLUSIONS: These results show that cspA and cspB expressions are differentially regulated at low temperature indicating that E. coli contains at least two different biothermostats or thermoregulators that are likely to play important roles in cellular adaptation to low temperature. The cspB promoter shows sequence similarity to the cspA promoter. Furthermore, both cspA and cspB mRNAs have unusually long 5' untranslated regions (159 and 161 bases, respectively), both of which are able to form similar extensive secondary structures. These features are considered to contribute to the nature of the thermostats for cspA and cspB.

• Graumann P, Schroder K, Schmid R, Marahiel MA
• Cold shock stress-induced proteins in Bacillus subtilis.
• J Bacteriol. 1996; 178: 4611-9
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• Bacteria respond to a decrease in temperature with the induction of proteins that are classified as cold-induced proteins (CIPs). Using two-dimensional gel electrophoresis, we analyzed the cold shock response in Bacillus subtilis. After a shift from 37 to 15 degrees C the synthesis of a majority of proteins was repressed; in contrast, 37 proteins were synthesized at rates higher than preshift rates. One hour after cold shock, the induction of CIPs decreased, and after 2 h, general protein synthesis resumed. The identified main CIPs were excised from two-dimensional gels and were subjected to microsequencing. Three small acidic proteins that showed the highest relative induction after cold shock were highly homologous and belonged to a protein family of which one member, the major cold shock protein, CspB, has previously been characterized. Two-dimensional gel analyses of a cspB null mutant revealed that CspB affects the level of induction of several CIPs. Other identified CIPs function at various levels of cellular physiology, such as chemotaxis (CheY), sugar uptake (Hpr), translation (ribosomal proteins S6 and L7/L12), protein folding (PPiB), and general metabolism (CysK, Ilvc, Gap, and triosephosphate isomerase).

• Ingham CJ, Hunter IS, Smith MC
• Isolation and sequencing of the rho gene from Streptomyces lividans ZX7 and characterization of the RNA-dependent NTPase activity of the overexpressed protein.
• J Biol Chem. 1996; 271: 21803-7
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• The gene for transcription termination factor Rho was isolated from Streptomyces lividans ZX7. It encoded a 77-kDa polypeptide (Rho 77) with considerable homology to known Rho factors. An atypical hydrophilic region of 228 residues was found within the N-terminal RNA-binding domain. Only Rho from Micrococcus luteus and Mycobacterium leprae (closely related GC-rich Gram-positive bacteria) had an analogous sequence. Rho 77 was overexpressed in Escherichia coli and purified using an N-terminal hexahistidine-tag. Rho 77 displayed a broad RNA-dependent ATPase activity, with poly(C) RNA being no more than 4-fold more effective than poly(A). This contrasts with the ATPase activity of Rho from E. coli which is stimulated primarily by poly(C) RNA. Rho 77 was a general RNA-dependent NTPase, apparent Km values for NTPs were: GTP 0.13 mM, ATP 0.17 mM, UTP 1.1 mM, and CTP >2 mM. Rho 77 poly(C)-dependent ATPase activity was inhibited by heparin, unlike the E. coli Rho. The antibiotic bicyclomycin inhibited the in vitro RNA-dependent ATPase activity of Rho 77, did not inhibit growth of streptomycetes but delayed the development of aerial mycelia. N-terminal deletion analysis to express a truncated form of Rho (Rho 72, 72 kDa) indicated that the first 42 residues of Rho 77 were not essential for RNA-dependent NTPase activity and were not the targets of inhibition by heparin or bicyclomycin.

• Manna AC, Pai KS, Bussiere DE, White SW, Bastia D
• The dimer-dimer interaction surface of the replication terminator protein of Bacillus subtilis and termination of DNA replication.
• Proc Natl Acad Sci U S A. 1996; 93: 3253-8
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• The replication terminator protein (RTP) of Bacillus subtilis causes polar fork arrest at replication termini by sequence-specific interaction of two dimeric proteins with the terminus sequence. The crystal structure of the RTP protein has been solved, and the structure has already provide valuable clues regarding the structural basis of its function. However, it provides little information as to the surface of the protein involved in dimer-dimer interaction. Using site-directed mutagenesis, we have identified three sites on the protein that appear to mediate the dimer-dimer interaction. Crystallographic analysis of one of the mutant proteins (Y88F) showed that its structure is unaltered when compared to the wild-type protein. The locations of the three sites suggested a model for the dimer-dimer interaction that involves an association between two beta-ribbon motifs. This model is supported by a fourth mutation that was predicted to disrupt the interaction and was shown to do so. Biochemical analyses of these mutants provide compelling evidence that cooperative protein-protein interaction between two dimers of RTP is essential to impose polar blocks to the elongation of both DNA and RNA chains.

• Antson AA et al.
• The structure of trp RNA-binding attenuation protein.
• Nature. 1995; 374: 693-700
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• The crystal structure of the trp RNA-binding attenuation protein of Bacclius subtilis solved at 1.8 A resolution reveals a novel structural arrangement in which the eleven subunits are stabilized through eleven intersubunit beta-sheets to form a beta-wheel with a large central hole. The nature of the binding of L-tryptophan in clefts between adjacent beta-sheets in the beta-wheel suggests that this binding induces conformational changes in the flexible residues 25-33 and 49-52. It is argued that upon binding, the messenger RNA target forms a matching circle in which eleven U/GAG repeats are bound to the surface of the protein ondecamer modified by the binding of L-tryptophan.

• Bussiere DE, Bastia D, White SW
• Crystal structure of the replication terminator protein from B. subtilis at 2.6 A.
• Cell. 1995; 80: 651-60
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• The crystal structure of the replication terminator protein (RTP) of B. subtilis has been determined at 2.6 A resolution. As previously suggested by both biochemical and biophysical studies, the molecule exists as a symmetric dimer and is in the alpha + beta protein-folding class. The protein has several uncommon features, including an antiparallel coiled-coil, which serves as the dimerization domain, and both an alpha-helix and a beta-ribbon suitably positioned to interact with the major and minor grooves of B-DNA. A site has been identified on the surface of RTP that is biochemically and positionally suitable for interaction with the replication-specific helicase. Other features of the structure are consistent with the polar contrahelicase mechanism of the protein. A model of the interaction between RTP and its cognate DNA is presented.

• Pereira S, Platt T
• A mutation in the ATP binding domain of rho alters its RNA binding properties and uncouples ATP hydrolysis from helicase activity.
• J Biol Chem. 1995; 270: 30401-7
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• The Escherichia coli mutant rho201 was originally isolated in a genetic screen for defects in rho-dependent termination. Cloning and sequencing of this gene reveals a single phenylalanine to cysteine mutation at residue 232 in the ATP binding domain of the protein. This mutation significantly alters its RNA binding properties so that it binds trp t', RNA 100-fold weaker than the wild type protein, with a Kd of approximately 1.3 nM. Rho201 binds nonspecific RNA only 3-4-fold less tightly than it binds trp t', while the wild type differential for these same RNAs is 10-20-fold. Curiously, rho201 displays increased secondary site RNA activation, with a Km for ribo(C)10 of 0.6 microM, compared to the wild type value of 3-4 microM. Although rho201 and the wild type protein hydrolyze ATP similarly with poly(C), or trp t' RNA, as cofactors, rho201 has a higher ATPase activity when activated by nonspecific RNA. Physically, rho201 displays an abnormal conformation detectable by mild trypsin digestion. Despite effective ATP hydrolysis, the rho201 mutant is a poor RNA:DNA helicase and terminates inefficiently on trp t'. The single F232C mutation thus appears to uncouple the protein's ATPase activity from its helicase function, so rho can no longer harness available energy for use in subsequent reactions.

• Beger RD, Balasubramanian S, Bennett SE, Mosbaugh DW, Bolton PH
• Tertiary structure of uracil-DNA glycosylase inhibitor protein.
• J Biol Chem. 1995; 270: 16840-7
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• The Bacillus subtilis bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) is an acidic protein of 84 amino acids that inactivates uracil-DNA glycosylase from diverse organisms. The secondary structure of Ugi consists of five anti-parallel beta-strands and two alpha-helices (Balasubramanian, S., Beger, R.D., Bennett, S.E., Mosbaugh, D.W., and Bolton, P.H. (1995) J. Biol. Chem. 270, 296-303). The tertiary structure of Ugi has been determined by solution state multidimensional nuclear magnetic resonance. The Ugi structure contains an area of highly negative electrostatic potential produced by the close proximity of a number of acidic residues. The unfavorable interactions between these acidic residues are apparently accommodated by the stability of the beta-strands. This negatively charged region is likely to play an important role in the binding of Ugi to uracil-DNA glycosylase.

• Miwa Y, Horiguchi T, Shigesada K
• Structural and functional dissections of transcription termination factor rho by random mutagenesis.
• J Mol Biol. 1995; 254: 815-37
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• Transcription termination factor rho from Escherichia coli is a homohexamer of 419 amino acid subunits and catalyzes an ATP-dependent release of nascent RNA transcripts. A rho monomer has three distinct domains functioning independently at the first approximation: the amino-terminal one quarter containing a primary RNA-binding site, the central 270-amino acids region constituting an ATP-binding domain with homologies to F1-ATPase, and the carboxy-terminal remainder with unknown function(s). To further delineate the structural and functional organizations of rho protein, we undertook its random mutagenesis using error-prone polymerase chain reactions with the carboxy-terminal 100-amino acid region chosen as the initial target. From 14 mutants identified, rho protein was purified and characterized in vitro. Of these, 11 mutants are defective in termination in vivo and show decreased activities in various partial functions examined: ATP binding; RNA binding; and ATPase activities dependent on three cofactors with decreasing efficacies, poly(C), lambda cro RNA and poly(U). A few of them are also affected in the putative secondary RNA-binding site that is functionally coupled to ATP hydrolysis. By contrast, the three other mutants are hyperactive in termination, poly(U)-dependent ATPase activity, and RNA interaction at the primary site. In these properties, the hyper-terminating mutants strikingly resemble the "super rho" mutant formerly found in the amino-terminal domain. Taken together, these findings indicate that the carboxy-terminal region plays a pivotal role in functionally coupling the RNA and ATP-binding domains, plausibly by acting as an interface for their interaction within or across individual subunits. In light of the reported X-ray crystallographic structure of F1-ATPase, we propose a model for the tertiary and quaternary structure of rho that is consistent with the observed mutational effects as well as a number of structural and functional properties characteristic of rho.

• Schroder K, Graumann P, Schnuchel A, Holak TA, Marahiel MA
• Mutational analysis of the putative nucleic acid-binding surface of the cold-shock domain, CspB, revealed an essential role of aromatic and basic residues in binding of single-stranded DNA containing the Y-box motif.
• Mol Microbiol. 1995; 16: 699-708
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• The major cold-shock protein of Bacillus subtilis, CspB, is a member of a protein family widespread among prokaryotes and eukaryotes that share the highly conserved cold-shock domain (CSD). The CSD domain is involved in transcriptional and translational regulation and was shown to bind the Y-box motif, a cis-element that contains the core sequence ATTGG, with high affinity. The three-dimensional structure of CspB, a prototype of this protein family, revealed that this hydrophilic CSD domain creates a surface rich in aromatic and basic amino acids that may act as the nucleic acid-binding site. We have analysed the potential role of conserved aromatic and basic residues in nucleic acid binding by site-directed mutagenesis. In gel retardation and ultraviolet cross-linking experiments, the ability of CspB mutants to bind single-stranded oligonucleotides (ssDNA) that contain the Y-box motif was investigated. Single substitutions of three highly conserved phenylalanine residues (Phe-15, Phe-17, Phe-27) by alanine and substitution of one histidine (His-29) by glutamine, all located within the putative RNA-binding sites RNP-1 and RNP-2, abolished the nucleic acid-binding activity of CspB. Conservative substitutions of Phe-15 to tyrosine (F15Y) showed a small increase in binding affinity, whereas separate replacement of Phe-17 and Phe-27 by tyrosine caused a reduction in binding activity. These and other substitutions including the conserved basic residues Lys-7, Lys-13 and Arg-56 as well as the aromatic residues Trp-8 and Phe-30 strongly suggest that CspB uses the side-chains of these amino acids for specific interaction with nucleic acids. Ultraviolet cross-linking experiments for CspB mutants with ssDNA supported the idea of specific CspB/nucleic acid interaction and indicated an essential role for the aromatic and basic residues in this binding. In addition, two-dimensional nuclear magnetic resonance studies with F17A, K13Q, F15Y and F27Y revealed that the mutants have the same overall structure as the wild-type CspB protein.

• Lottering EA, Streips UN
• Induction of cold shock proteins in Bacillus subtilis.
• Curr Microbiol. 1995; 30: 193-9
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• The cold shock response in the Gram-positive soil bacterium Bacillus subtilis is described. Cells were exposed to sudden decreases in temperature from their optimal growth temperature of 37 degrees C. The B. subtilis cells were cold shocked at 25 degrees C, 20 degrees C, 15 degrees C, and 10 degrees C. A total of 53 polypeptides were induced at the various cold shock temperatures and were revealed by two-dimensional gel electrophoresis. General stress proteins were identified by a comparative analysis with the heat shock response of B. subtilis. Some unique, prominent cold shock proteins such as the 115 kDa, 97 kDa, and 21 kDa polypeptides were microsequenced. Sequence comparison demonstrated that the 115-kDa protein had homology to the TCA cycle enzyme, aconitase.

• Folmer RH, Nilges M, Konings RN, Hilbers CW
• Solution structure of the single-stranded DNA binding protein of the filamentous Pseudomonas phage Pf3: similarity to other proteins binding to single-stranded nucleic acids.
• EMBO J. 1995; 14: 4132-42
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• The three-dimensional structure of the homodimeric single-stranded DNA binding protein encoded by the filamentous Pseudomonas bacteriophage Pf3 has been determined using heteronuclear multidimensional NMR techniques and restrained molecular dynamics. NMR experiments and structure calculations have been performed on a mutant protein (Phe36 --> His) that was successfully designed to reduce the tendency of the protein to aggregate. The protein monomer is composed of a five-stranded antiparallel beta-sheet from which two beta-hairpins and a large loop protrude. The structure is compared with the single-stranded DNA binding protein encoded by the filamentous Escherichia coli phage Ff, a protein with a similar biological function and DNA binding properties, yet quite different amino acid sequence, and with the major cold shock protein of Escherichia coli, a single-stranded DNA binding protein with an entirely different sequence, biological function and binding characteristics. The amino acid sequence of the latter is highly homologous to the nucleic acid binding domain (i.e. the cold shock domain) of proteins belonging to the Y-box family. Despite their differences in amino acid sequence and function, the folds of the three proteins are remarkably similar, suggesting that this is a preferred folding pattern shared by many single-stranded DNA binding proteins.

• Pereira S, Platt T
• Analysis of E. coli rho factor: mutations affecting secondary-site interactions.
• J Mol Biol. 1995; 251: 30-40
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• To define and differentiate primary and secondary RNA binding sites within the linear sequence of the rho protein, we investigated two mutant alleles, rho-115 and rhosuA1. They were first identified as defective in transcription termination in vivo, and later demonstrated to be defective in their interactions with RNA at the primary and secondary sites, respectively. Sequencing of rhosuA1 revealed a single lysine to glutamic acid residue change at position 352 (KE352), while rho-115 carries two mutations, glycine99 to valine (GV99) and a proline235 to histidine (PH235). Proteins carrying single mutations at each of these three positions were purified and their characteristics compared to the wild-type protein. We found both KE352 and GV99 to be defective in secondary-site RNA activation, with Km values for r(C)10 of 100 microM and approximately 650 microM, respectively, compared to the wild-type value of 4 microM. These observed secondary-site defects correlated with decreased helicase and ATPase activities, as well as a loss of transcription termination activity in vitro. By contrast, PH235 was very efficient at interacting with r(C)10 at the secondary site, with a measured Km of 0.5 microM, and displayed the characteristics of a hyperactive rho, as judged by its ATPase, helicase and termination capabilities. Our results show that mutations at three very different locations in the polypeptide can affect secondary-site activation by RNA, and that these interactions play a pivotal role in ATP hydrolysis, helicase activity and transcription termination.

• Yu L, Zhu CX, Tse-Dinh YC, Fesik SW
• Solution structure of the C-terminal single-stranded DNA-binding domain of Escherichia coli topoisomerase I.
• Biochemistry. 1995; 34: 7622-8
• Display abstract

• Escherichia coli DNA topoisomerase I catalyzes the interconversion of different topological forms of DNA. In this paper we describe NMR studies of a 14K C-terminal fragment of this enzyme that binds preferentially to single-stranded DNA and enhances the enzyme's ability to relax negatively supercoiled DNA under high salt conditions. The 1H, 13C, and 15N resonances of the protein were assigned from a number of heteronuclear multidimensional NMR experiments, and the three-dimensional structure of the protein was determined from a total of 2188 NMR-derived restraints. The root-mean-square deviation about the mean coordinate positions for residues 13-120 is 0.68 +/- 0.11 A for the backbone atoms and 1.09 +/- 0.09 A for all heavy atoms. The overall fold, which consists of two four-stranded beta-sheets separated by two helices, differs from other DNA- and RNA-binding proteins such as gene 5, cold shock protein, and hnRNP C. From an analysis of the changes in chemical shift upon the addition of single-stranded DNA, the location of the oligonucleotide binding site was determined. The binding site consists of a beta-sheet containing positively charged and aromatic amino acids and, in spite of its different structure, is similar to that found in other proteins that bind single-stranded oligonucleotides.

• Makhatadze GI, Marahiel MA
• Effect of pH and phosphate ions on self-association properties of the major cold-shock protein from Bacillus subtilis.
• Protein Sci. 1994; 3: 2144-7
• Display abstract

• The intermolecular interactions of the major cold-shock protein from Bacillus subtilis (CspB) in solution in the presence of different salts, including phosphate, have been studied by means of scanning calorimetry and size-exclusion chromatography. Calorimetric results indicate that, in all cases, protein unfolding can be approximated by a 2-state model, but the modes of unfolding can differ depending on the conditions. In the presence of phosphate, the cooperative folding unit is a monomer, whereas in the absence of phosphate, the cooperative unit is a dimer. The difference in the self-association of CspB in the presence and absence of phosphate was supported by size-exclusion chromatography. These results are compared with recent structural studies of CspB in crystal and in solution.

• Murray MT
• Nucleic acid-binding properties of the Xenopus oocyte Y box protein mRNP3+4.
• Biochemistry. 1994; 33: 13910-7
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• Y box proteins contain the conserved cold shock domain (CSD) and several basic/aromatic (B/A) islands that are rich in arginine and aromatic residues. The binding of purified Xenopus oocyte 6S Y box protein, mRNP3+4, to Y box RNA, single-stranded (ss) DNA, and double-stranded (ds) DNA was studied by gel mobility shift and nitrocellulose filter binding assays. mRNP3+4 specifically bound Y box ssDNA or RNA, while binding of dsDNA was not detected. Y box ssDNA and RNA did not efficiently cross-compete for mRNP3+4 binding, and no evidence for ternary complex formation was detected. However, Y box ssDNA binding was competed by high concentrations of Y box RNA or nonspecific RNA competitors, indicating that the ssDNA-binding site has a lower affinity for RNA. mRNP3+4 demonstrated similar affinity for either Y box RNA or ssDNA. However, at elevated ionic strength RNA binding was markedly greater than ssDNA binding, indicating that RNA binding involves nonionic interactions that are not utilized for ssDNA binding. Recombinant polypeptides containing B/A islands bound Y box RNA exclusively, but inclusion of the CSD led to preferential ssDNA binding. The results demonstrate that the B/A islands are exclusively RNA-binding, while the CSD exhibits preferential binding of ssDNA. The inability of Y box RNA and ssDNA to efficiently cross-compete for mRNP3+4 binding suggests that isoforms exhibit preferential ssDNA or RNA binding.

• Schindelin H, Jiang W, Inouye M, Heinemann U
• Crystal structure of CspA, the major cold shock protein of Escherichia coli.
• Proc Natl Acad Sci U S A. 1994; 91: 5119-23
• Display abstract

• The major cold shock protein of Escherichia coli, CspA, produced upon a rapid downshift in growth temperature, is involved in the transcriptional regulation of at least two genes. The protein shares high homology with the nucleic acid-binding domain of the Y-box factors, a family of eukaryotic proteins involved in transcriptional and translational regulation. The crystal structure of CspA has been determined at 2-A resolution and refined to R = 0.187. CspA is composed of five antiparallel beta-strands forming a closed five-stranded beta-barrel. The three-dimensional structure of CspA is similar to that of the major cold shock protein of Bacillus subtilis, CspB, which has recently been determined at 2.45-A resolution. However, in contrast to CspB, no dimer is formed in the crystal. The surface of CspA is characteristic for a protein interacting with single-stranded nucleic acids. Due to the high homology of the bacterial cold shock proteins with the Y-box factors, E. coli CspA and B. subtilis CspB define a structural framework for the common cold shock domain.

• Modrak D, Richardson JP
• The RNA-binding domain of transcription termination factor rho: isolation, characterization, and determination of sequence limits.
• Biochemistry. 1994; 33: 8292-9
• Display abstract

• The function of transcription termination factor rho from Escherichia coli is dependent upon its ability to bind RNA. To delineate the extent of the RNA-binding domain in the rho polypeptide, plasmid-borne copies of altered forms of the rho gene were expressed to yield truncated versions. These proteins were then isolated and assayed for their ability to bind an RNA oligonucleotide [oligo(C)8] using an ultraviolet light-induced cross-linking assay. A fragment consisting of the first 116 amino acid residues, rho(1-116), bound oligo(C)8 with nearly the same affinity and specificity as the intact protein. Smaller derivatives lacking 5, 13, or 22 residues from the N terminus or with 2 fewer residues at the C terminus bound RNA with reduced affinity, while derivatives lacking 27 N-terminal residues or having just the first 109 residues were unable to bind RNA. Derivatives lacking N-terminal residues were considerably less soluble than rho(1-116). The physical properties of rho(1-116) indicate that it possesses approximately 20% each of alpha-helix and beta-sheet and is monomeric in solution. Thus, the results show that this fragment, which contains an RNP1 sequence motif, will be a good model for future physical-chemical studies of the protein-RNA interactions of rho.

• Steinmetz EJ, Platt T
• Evidence supporting a tethered tracking model for helicase activity of Escherichia coli Rho factor.
• Proc Natl Acad Sci U S A. 1994; 91: 1401-5
• Display abstract

• Transcription termination factor Rho of Escherichia coli has an ATP-dependent RNA.DNA helicase activity that presumably facilitates RNA transcript release from the elongation complex. This helicase activity is unidirectional (5' to 3') and is stoichiometric, with one RNA molecule released per Rho hexamer in vitro. A simple RNA tracking model postulates that after Rho's initial binding, it translocates preferentially toward the 3' end of the RNA. Nitrocellulose filter binding studies combined with RNase H cleavage are inconsistent with this simple tracking model. Instead, they support a model in which Rho forms tight primary binding interactions with the recognition region of the RNA and remains bound there while transient secondary RNA binding interactions coupled to ATP hydrolysis serve to scan along the RNA to contact the RNA.DNA helix. This "tethered tracking" model is consistent with other properties of Rho factor, including the presence of two classes of RNA binding sites on the Rho hexamer and the 1:1 stoichiometry in the Rho helicase assay.

• O I, Stitt BL
• 8-Azido-ATP inactivation of Escherichia coli transcription termination factor Rho. Modification of one subunit inactivates the hexamer.
• J Biol Chem. 1994; 269: 5009-15
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• Escherichia coli transcription termination factor Rho (EC 3.6.1.3) releases nascent RNA from transcription complexes in a reaction which requires ATP hydrolysis. To understand the structure of the ATPase active site, we employed an analog of ATP, 8-azidoadenosine 5'-triphosphate (8-azido-ATP) as a photoaffinity labeling agent. 8-Azido-ATP interacts nearly normally with the active site of Rho. It binds to 3 sites per Rho hexamer with a 100 microM KD and is a substrate with a Vmax 5% that of ATP and a Km of 18 microM. Under UV irradiation, 8-azido-ATP makes covalent bonds with Rho, inactivating its ATPase. Rho is protected from this inactivation by the presence of ATP. We used [alpha-32P]8-azido-ATP to label the active site and identify residues involved in ATP binding. Labeled tryptic peptides of the modified Rho were purified by Fe(3+)-iminodiacetic acid affinity chromatography and reverse-phase C18 column high performance liquid chromatography. We identified a single peptide, Gly174-Lys184, that is labeled by 8-azido-ATP and protected from labeling in the presence of ATP. The modified amino acid is Lys181, whose conservative replacement by Gln181 gives rise to a poorly active enzyme (Dombroski, A. J., Brennan, C. A., Spear, P., and Platt, T. (1988a) J. Biol. Chem. 263, 18802-18809). Lys181 probably participates in binding the phosphoryl groups of ATP. Incorporation of one 8-azido-ATP per Rho hexamer is sufficient to cause inactivation, a result that indicates that the active sites of Rho interact in RNA-dependent ATP hydrolysis.

• Ladomery M, Sommerville J
• Binding of Y-box proteins to RNA: involvement of different protein domains.
• Nucleic Acids Res. 1994; 22: 5582-9
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• Eukaryotic Y-box proteins are reported to interact with a wide variety of nucleic acid structures to act as transcription factors and mRNA masking proteins. The modular structure of Y-box proteins includes a highly conserved N-terminal cold-shock domain (CSD, equivalent to the bacterial cold-shock proteins) plus four basic C-terminal domains containing arginine clusters and aromatic residues. In addition, the basic domains are separated by acidic regions which contain several potential sites for serine/threonine phosphorylation. The interaction of Y-box proteins, isolated from Xenopus oocytes (FRGY2 type), with RNA molecules has been studied by UV crosslinking and protein fragmentation. We have identified two distinct binding activities. The CSD interacts preferentially with the polypurines poly(A,G) and poly(G) but not poly(A), this activity being sensitive to 5 mM MgCl2 but not to 5 mM spermidine. In the presence of 1 mM MgCl2 or 1 mM spermidine, the basic domains interact preferentially with poly(C,U), this activity being sensitive to 0.5 M NaCl. Binding of the basic domains is also sensitive to low concentrations of heparin. The basic domains can be crosslinked individually to labelled RNA. These results are discussed with reference to the various specificities noted in the binding of Y-box proteins to RNA and DNA.

• Oubridge C, Ito N, Evans PR, Teo CH, Nagai K
• Crystal structure at 1.92 A resolution of the RNA-binding domain of the U1A spliceosomal protein complexed with an RNA hairpin.
• Nature. 1994; 372: 432-8
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• The crystal structure of the RNA-binding domain of the small nuclear ribonucleoprotein U1A bound to a 21-nucleotide RNA hairpin has been determined at 1.92 A resolution. The ten-nucleotide RNA loop binds to the surface of the beta-sheet as an open structure, and the AUUGCAC sequence of the loop interacts extensively with the conserved RNP1 and RNP2 motifs and the C-terminal extension of the RNP domain. These interactions include stacking of RNA bases with aromatic side chains of proteins and many direct and water-mediated hydrogen bonds. The structure reveals the stereochemical basis for sequence-specific RNA recognition by the RNP domain.

• Graumann P, Marahiel MA
• The major cold shock protein of Bacillus subtilis CspB binds with high affinity to the ATTGG- and CCAAT sequences in single stranded oligonucleotides.
• FEBS Lett. 1994; 338: 157-60
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• We have characterized the nucleic acid binding properties of the major cold shock protein of Bacillus subtilis, CspB. CspB is a member of the cold shock domain (CSD) family, which is widespread among pro- and eukaryotes and shares the nucleic acid binding domain CSD. The CSD domain is highly conserved and binds with strong affinity to the Y-box motif, a cis-element that contains the CTGATTGGC/TC/TAA sequence. In a series of gel retardation experiments using oligonucleotides, which contain the Y-box motif and altered sequences, we show the preferential binding of CspB to single-stranded DNA that contains the ATTGG as well as the complementary CCAAT Y-box core sequence. In contrast CspB exhibits lower affinity to altered Y-box core sequences. Dependent on the length of the oligonucleotide and the degree of sequence deviation from the Y-box core sequence 3- to over 10-fold overexcess of CspB was needed for complete retardation.

• Opperman T, Richardson JP
• Phylogenetic analysis of sequences from diverse bacteria with homology to the Escherichia coli rho gene.
• J Bacteriol. 1994; 176: 5033-43
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• Genes from Pseudomonas fluorescens, Chromatium vinosum, Micrococcus luteus, Deinococcus radiodurans, and Thermotoga maritima with homology to the Escherichia coli rho gene were cloned and sequenced, and their sequences were compared with other available sequences. The species for all of the compared sequences are members of five bacterial phyla, including Thermotogales, the most deeply diverged phylum. This suggests that a rho-like gene is ubiquitous in the Bacteria and was present in their common ancestor. The comparative analysis revealed that the Rho homologs are highly conserved, exhibiting a minimum identity of 50% of their amino acid residues in pairwise comparisons. The ATP-binding domain had a particularly high degree of conservation, consisting of some blocks with sequences of residues that are very similar to segments of the alpha and beta subunits of F1-ATPase and of other blocks with sequences that are unique to Rho. The RNA-binding domain is more diverged than the ATP-binding domain. However, one of its most highly conserved segments includes a RNP1-like sequence, which is known to be involved in RNA binding. Overall, the degree of similarity is lowest in the first 50 residues (the first half of the RNA-binding domain), in the putative connector region between the RNA-binding and the ATP-binding domains, and in the last 50 residues of the polypeptide. Since functionally defective mutants for E. coli Rho exist in all three of these segments, they represent important parts of Rho that have undergone adaptive evolution.

• Wolffe AP
• Structural and functional properties of the evolutionarily ancient Y-box family of nucleic acid binding proteins.
• Bioessays. 1994; 16: 245-51
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• The Y-box proteins are the most evolutionarily conserved nucleic acid binding proteins yet defined in bacteria, plants and animals. The central nucleic acid binding domain of the vertebrate proteins is 43% identical to a 70-amino-acid-long protein (CS7.4) from E. coli. The structure of this domain consists of an antiparallel five-stranded beta-barrel that recognizes both DNA and RNA. The diverse biological roles of these Y-box proteins range from the control of the E. coli cold-shock stress response to the translational masking of messenger RNA in vertebrate gametes. This review discusses the organization of the prokaryotic and eukaryotic Y-box proteins, how they interact with nucleic acids, and their biological roles, both proven and potential.

• Wang Y, von Hippel PH
• Escherichia coli transcription termination factor rho. II. Binding of oligonucleotide cofactors.
• J Biol Chem. 1993; 268: 13947-55
• Display abstract

• The relative binding affinities for rho of the oligonucleotide rho ATPase cofactors studied in the accompanying paper (Wang, Y., and von Hippel, P. H. (1993) J. Biol. Chem. 268, 13940-13946) have been determined by gel mobility shift and ultrafiltration binding analyses. We find that each rho hexamer carries three strong and three weak RNA-binding sites that differ approximately 10-fold in their affinities for oligonucleotide cofactors. Furthermore, in contrast to the sequence dependence of ATPase activation, we find that the binding affinities of these oligonucleotide cofactors for rho depend only on their cytosine content. In addition, we show that changes in the positions of rU residues in the oligo(rU,rC) cofactors (which significantly modulate the ATPase activity of rho) have no effect on binding affinities and that the addition of ATP, ADP, or the nonhydrolyzable ATP analog adenosine 5'-(beta,gamma-methylene)triphosphate also does not change the binding affinities of the oligonucleotide cofactors for rho. Considered in the context of the coupling of the rho ATPase and RNA binding and release cycles, these results suggest that rC residues are required for the formation of stable rho-RNA complexes, whereas rU residues at the 5' termini of cofactors bound to rho initiate or facilitate the release of the RNA from the individual cofactor site as a consequence of ATP hydrolysis. Thus, both the tightness of the binding of RNA segments to the individual RNA-binding sites of rho and the rate of release of these segments from these sites are critical in controlling the ATPase rate of rho and probably also in modulating the function of this protein in transcript termination.

• Geiselmann J, Wang Y, Seifried SE, von Hippel PH
• A physical model for the translocation and helicase activities of Escherichia coli transcription termination protein Rho.
• Proc Natl Acad Sci U S A. 1993; 90: 7754-8
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• Transcription termination protein Rho of Escherichia coli interacts with newly synthesized RNA chains and brings about their release from elongation complexes paused at specific Rho-dependent termination sites. Rho is thought to accomplish this by binding to a specific Rho "loading site" on the nascent RNA and then translocating preferentially along the transcript in a 5'-->3' direction. On reaching the elongation complex, Rho releases the nascent RNA by a 5'-->3' RNA.DNA helicase activity. These translocation and helicase activities are driven by the RNA-dependent ATPase activity of Rho. In this paper we propose a mechanism for these processes that is based on the structure and properties of the Rho protein. Rho is a hexamer of identical subunits that are arranged as a trimer of asymmetric dimers with D3 symmetry. The binding of ATP and RNA to Rho also reflects this pattern; the Rho hexamer carries three strong and three weak binding sites for each of these entities. The asymmetric dimers of Rho correspond to functional dimers that can undergo conformational transitions driven by ATP hydrolysis. We propose that the quaternary structure of Rho coordinates the ATP-driven RNA binding and release processes to produce a biased random walk of the Rho hexamer along the RNA, followed by RNA.DNA helicase activity and transcript release. The proposed model may have implications for other hexameric DNA.DNA, RNA.DNA, and RNA.RNA helicases that function in replication and transcription.

• Golden BL, Ramakrishnan V, White SW
• Ribosomal protein L6: structural evidence of gene duplication from a primitive RNA binding protein.
• EMBO J. 1993; 12: 4901-8
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• In all cells, protein synthesis is coordinated by the ribosome, a large ribonucleoprotein particle that is composed of > 50 distinct protein molecules and several large RNA molecules. Here we present the crystal structure of ribosomal protein L6 from the thermophilic bacterium Bacillus stearothermophilus solved at 2.6 A resolution. L6 contains two domains with almost identical folds, implying that it was created by an ancient gene duplication event. The surface of the molecule displays several likely sites of interaction with other components of the ribosome. The RNA binding sites appear to be localized in the C-terminal domain whereas the N-terminal domain contains the potential sites for protein-protein interactions. The domain structure is homologous with several other ribosomal proteins and to a large family of eukaryotic RNA binding proteins.

• Gibson TJ, Thompson JD, Heringa J
• The KH domain occurs in a diverse set of RNA-binding proteins that include the antiterminator NusA and is probably involved in binding to nucleic acid.
• FEBS Lett. 1993; 324: 361-6
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• New findings are presented for the approximately 50 residue KH motif, a domain recently discovered in RNA-binding proteins. The conserved sequence is approximately 10 residues larger than previously reported. Profile searches have revealed new members of this family, including two, E. coli NusA and human GAP-associated p62 phosphoprotein, for which RNA-binding data exists. A nusA homolog was detected in the RNA polymerase gene complex of six archaebacterial species and may encode an antiterminator. All KH-containing proteins are linked with RNA and the KH motif most probably functions as a nucleic acid binding domain.

• Schroder K, Zuber P, Willimsky G, Wagner B, Marahiel MA
• Mapping of the Bacillus subtilis cspB gene and cloning of its homologs in thermophilic, mesophilic and psychrotrophic bacilli.
• Gene. 1993; 136: 277-80
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• The Bacillus subtilis cold shock (CS)-inducible gene, cspB, encoding the nucleic-acid-binding, major CS protein CspB, is located at about 80 degrees on the B. subtilis genetic map. Using this cspB as a probe, the CspB-encoding genes from two thermophilic bacilli were cloned and characterized. The nucleotide (nt) sequences of the B. caldolyticus and B. stearothermophilus cspB coding regions are 78 and 76% identical to the B. subtilis cspB and the deduced amino acid (aa) sequences revealed 84 and 82% identity, respectively. The cspB genes of the mesophilic B. globigii and the some what psychrotrophic B. globisporus, were amplified by PCR using mixed degenerate oligodeoxyribonts based on the 5' and 3' ends of B. subtilis cspB. The nt sequence comparisons of the resulting cloned PCR fragments revealed 98 to 99% identity to cspB of B. subtilis and 97% aa identity to the CspB protein. The high conservation of CspB within the genus Bacillus and the presence of a related nucleic acid-binding domain within several eukaryotic transcription factors implies an important common biological function that seems to be highly conserved from bacteria to man.

• Wang Y, von Hippel PH
• Escherichia coli transcription termination factor rho. I. ATPase activation by oligonucleotide cofactors.
• J Biol Chem. 1993; 268: 13940-6
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• Rho protein is required to bring about RNA release from Escherichia coli transcription complexes paused at specific (rho-dependent) termination sites. Rho functions in termination as a hexamer of identical subunits arranged in D3 symmetry, with each rho subunit carrying an RNA- and an ATP-binding site. The detailed mechanism of rho-catalyzed transcript release remains to be determined, but it is clear that the RNA-dependent ATPase activity that is stimulated by interaction with the nascent transcript is essential to the termination function of rho. In this study, we have used short (8-10 nucleotide residues) synthetic ribo-oligonucleotides to model the interaction of segments of the RNA cofactor with rho. A poly(dC) enhancement procedure was used to permit the measurement of steady state ATPase parameters. We show that (i) ATPase activation is cofactor composition- and sequence-dependent; (ii) at least 60% of the residues of these short RNA cofactors must be cytosine to produce maximal rho ATPase activation; (iii) oligo(rU,rC) cofactors with the rU residues located at the 5' termini of the oligomer are much better ATPase cofactors than oligomers containing rC residues only; (iv) this enhanced stimulation is not observed if the rU residues are replaced by rA residues; (v) this cofactor activity relative to oligo(rC) is reversed if the rU residues are placed at the 3' terminus of RNA oligomer; and (vi) these nucleotide sequence and composition effects do not appear to be functions of K+ or Mg2+ concentration. These ATPase activation results are correlated with the binding to rho of oligonucleotide cofactors in the accompanying paper (Wang, Y., and von Hippel, P. H. (1993) J. Biol. Chem. 268, 13947-13955).

• Geiselmann J, Seifried SE, Yager TD, Liang C, von Hippel PH
• Physical properties of the Escherichia coli transcription termination factor rho. 2. Quaternary structure of the rho hexamer.
• Biochemistry. 1992; 31: 121-32
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• Under approximately physiological conditions, the transcription termination factor rho from Escherichia coli is a hexamer of planar hexagonal geometry [Geiselmann, J., Yager, T. D., Gill, S. C., Calmettes, P., & von Hippel, P. H. (1992) Biochemistry (preceding paper in this issue)]. Here we describe studies that further define the quaternary structure of this hexamer. We use a combination of chemical cross-linking and treatment with mild denaturants to show that the fundamental unit within the rho hexamer is a dimer stabilized by an isologous (or pseudoisologous) bonding interface. Three identical dimers of rho interact via a second type of isologous bonding interface to yield a hexamer with C3 or D3 symmetry. Cross-linking and denaturation experiments definitely rule out C6 and C2 symmetry for the rho hexamer. Data from fluorescence quenching, lifetime, and energy transfer experiments also argue against C2 symmetry. The simplest symmetry assignment that is not contradicted by any experimental data is D3; thus we conclude that the rho hexamer has D3 symmetry. We also consider the positioning of the binding sites for RNA and ATP relative to the coordinate reference frame of the D3 hexamer. Fluorescence energy transfer data are presented and integrated with data from the literature to arrive at a self-consistent model for the quaternary structure of the rho hexamer.

• Geiselmann J, von Hippel PH
• Functional interactions of ligand cofactors with Escherichia coli transcription termination factor rho. I. Binding of ATP.
• Protein Sci. 1992; 1: 850-60
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• Escherichia coli transcription termination factor rho is an RNA-dependent ATPase, and ATPase activity is required for all its functions. We have characterized the binding of ATP to the physiologically relevant hexameric association state of rho in the absence of RNA and have shown that there are six ATP binding sites per rho hexamer. This stoichiometry has been verified by a number of different techniques, including ultracentrifugation, ultrafiltration, and fluorescence titration studies. We have also shown that ATP can bind to isolated monomers of rho when the hexamer is dissociated with the mild denaturant myristyltrimethylammonium bromide, demonstrating that each promoter of rho carries an ATP binding site. The six binding sites that we observe in the rho hexamer are not equivalent; the hexamer contains three strong (Ka approximately 3 x 10(6) M-1) and three weak (Ka approximately 10(5) M-1) binding sites for ATP. The binding constant of the weak binding site is just the reciprocal of the enzymatic Km for ATP as a substrate; thus these weak sites, as well as the strong sites, can, in principle, take part in the catalytic cycle. The asymmetry induced (or manifested) by ATP binding reduces the symmetry of the rho hexamer from a D3 to a pseudo-D3 state. This "breakage" of symmetry has implications for the molecular mechanism of rho, because an asymmetric structure can lead to directional helicase activity by invoking directionally distinct RNA binding and release reactions (see Geiselmann, J., Yager, T.D., & von Hippel, P.H., 1992c, Protein Sci. 1, 861-873).

• Geiselmann J, Yager TD, von Hippel PH
• Functional interactions of ligand cofactors with Escherichia coli transcription termination factor rho. II. Binding of RNA.
• Protein Sci. 1992; 1: 861-73
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• The rho protein of Escherichia coli interacts with the nascent RNA transcript while RNA polymerase is paused at specific rho-dependent termination sites on the DNA template, and (in a series of steps that are still largely undefined) brings about transcript termination at these sites. In this paper we characterize the interactions of rho with RNA and relate these interactions to the quaternary structure of the functional form of rho. We use CD spectroscopy and analytical ultracentrifugation to determine the binding interactions of rho with RNA ligands of defined length ([rC]n where n > or = 6). Rho binds to long RNA chains as a hexamer characterized by D3 symmetry. Each hexamer binds approximately 70 residues of RNA. We show by ultracentrifugation and dynamic laser light scattering that, in the presence of RNA ligands less than 22 nucleotide residues in length, rho changes its quaternary structure and becomes a homogeneous dodecamer. The dodecamer contains six strong binding sites for short RNA ligands: i.e., one site for every two rho protomers. The measured association constant of these short RNAs to rho increases with increasing (rC)n length, up to n = 9, suggesting that the binding site of each rho protomer interacts with 9 RNA nucleotide residues. Oligo (rC) ligands bound to the strong RNA binding sites on the rho dodecamer do not significantly stimulate the RNA-dependent ATPase activity of rho. Based on these features of the rho-RNA interaction and other experimental data we propose a molecular model of the interaction of rho with its cofactors.

• Willimsky G, Bang H, Fischer G, Marahiel MA
• Characterization of cspB, a Bacillus subtilis inducible cold shock gene affecting cell viability at low temperatures.
• J Bacteriol. 1992; 174: 6326-35
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• A new class of cold shock-induced proteins that may be involved in an adaptive process required for cell viability at low temperatures or may function as antifreeze proteins in Escherichia coli and Saccharomyces cerevisiae has been identified. We purified a small Bacillus subtilis cold shock protein (CspB) and determined its amino-terminal sequence. By using mixed degenerate oligonucleotides, the corresponding gene (cspB) was cloned on two overlapping fragments of 5 and 6 kb. The gene encodes an acidic 67-amino-acid protein (pI 4.31) with a predicted molecular mass of 7,365 Da. Nucleotide and deduced amino acid sequence comparisons revealed 61% identity to the major cold shock protein of E. coli and 43% identity to a family of eukaryotic DNA binding proteins. Northern RNA blot and primer extension studies indicated the presence of one cspB transcript that was initiated 119 bp upstream of the initiation codon and was found to be induced severalfold when exponentially growing B. subtilis cell cultures were transferred from 37 degrees C to 10 degrees C. Consistent with this cold shock induction of cspB mRNA, a six- to eightfold induction of a cspB-directed beta-galactosidase synthesis was observed upon downshift in temperature. To investigate the function of CspB, we inactivated the cold shock protein by replacing the cspB gene in the B. subtilis chromosome with a cat-interrupted copy (cspB::cat) by marker replacement recombination. The viability of cells of this mutant strain, GW1, at freezing temperatures was strongly affected. However, the effect of having no CspB in GW1 could be slightly compensated for when cells were preincubated at 10 degrees C before freezing. These results indicate that CspB belongs to a new type of stress-inducible proteins that might be able to protect B. subtilis cells from damage caused by ice crystal formation during freezing.

• Geiselmann J, Yager TD, Gill SC, Calmettes P, von Hippel PH
• Physical properties of the Escherichia coli transcription termination factor rho. 1. Association states and geometry of the rho hexamer.
• Biochemistry. 1992; 31: 111-21
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• To function as a DNA-RNA helicase in rho-dependent transcript termination, six genetically identical subunits of the Escherichia coli transcription termination protein rho must first assemble into a hexameric complex. To help determine the quaternary structure of this complex, we have studied the association equilibria of the rho protomers. Sedimentation equilibrium, sedimentation velocity, diffusion, X-ray scattering, and neutron-scattering data have been combined to create a "phase diagram" of the association states of this protein as a function of protein concentration and ionic environment. The results show that rho exists predominantly as a hexamer under approximately physiological conditions and that this hexamer is in equilibrium with both lower and higher states of association that may also have physiological relevance. Small-angle X-ray scattering measurements and theoretical calculations indicate that the rho hexamer has a radius of gyration of 50 +/- 3 A. The radius of gyration measured by small-angle neutron scattering in 2H2O is 47 +/- 3 A. These scattering studies also support earlier models of rho as a planar hexagon which have been developed on the basis of electron microscopy. In the following paper in this issue [Geiselmann, J., Seifried, S. E., Yager, T. D., Liang, C., & von Hippel, P. H. (1992)], these results are combined with information on symmetry, subunit interactions, and packing geometry to obtain a model of the quaternary structure of the functional rho hexamer.

• Brennan CA, Platt T
• Mutations in an RNP1 consensus sequence of Rho protein reduce RNA binding affinity but facilitate helicase turnover.
• J Biol Chem. 1991; 266: 17296-305
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• Escherichia coli rho protein facilitates transcription termination by a mechanism that involves rho binding to the nascent RNA, activation of rho's RNA-dependent ATPase activity, and release of the mRNA from the DNA template. The initial step, formation of a rho-RNA complex, is mediated primarily by an RNA binding domain included within the amino-terminal 151 amino acids of rho protein. We have now identified one specific portion of this region that is involved in RNA binding, by photocross-linking and by site-directed mutagenesis. UV irradiation of rho-RNA complexes results in covalent attachment of the RNA to a single peptide in rho that apparently spans amino acids 45-100. Within this peptide is a ribonucleoprotein (RNP1) consensus sequence, Gly-Phe-Gly-Phe, that is present in many RNA-binding proteins. Mutagenesis of the phenylalanine residues in this consensus to leucine or alanine results in mutant proteins that are defective for RNA binding and have altered ATPase and RNA-DNA helicase activities. The weakened affinity but increased salt sensitivity of RNA binding by the mutant proteins suggests that they have lost more than just a set of nonionic interactions and are consistent with a change in the conformation of the RNA binding site. Whatever the changes, they appear localized primarily to the RNA binding domain because the mutants retain much of their RNA-dependent ATPase activity. We infer that the Phe residues themselves do not play a substantial role in the activation of ATP hydrolysis. Our results indicate that several different components of RNA interaction are required for rho activity and support a role for the RNP1 consensus region of rho in at least one specific aspect of RNA binding.

• Gogol EP, Seifried SE, von Hippel PH
• Structure and assembly of the Escherichia coli transcription termination factor rho and its interaction with RNA. I. Cryoelectron microscopic studies.
• J Mol Biol. 1991; 221: 1127-38
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• Cryoelectron microscopy has been used to visualize the Escherichia coli transcription termination protein rho in a vitreously frozen state, without the use of strains, fixatives or other chemical perturbants. In the absence of RNA cofactor, a variety of structures are observed, reflecting the heterogeneity of complexes formed by rho at protein concentrations near the physiological range (3 to 10 microM). One of the most common structural motifs we see is a six-membered ring of rho subunits (present as either a closed or "notched" circle), which corresponds to the predominant hexameric association state of the protein. Also visible are smaller oligomeric structures, present as curved lines of rho subunits, which probably represent the lower association states of the protein that coexist with the hexamer at these protein concentrations. Addition of oligomers of ribocytosine (rC) of defined lengths (23-mers and 100-mers) results in the generation of more homogeneous populations of rho oligomers. In the presence of (rC)23, all identifiable particles appear either as closed or as notched hexameric circles. A small fraction of these particles are of visibly higher density, and are identified with the dodecamers expected as a subpopulation of rho under these conditions. Binding of (rC)100, an oligomer of length greater than that needed to span the entire hexamer binding site, results in a uniform population of closed circular hexamers. In some images additional features are visible at either the centers or the peripheries of the particles. These features may correspond to the excess length of the rC strands bound to the hexamers. The distributions of particles observed under the various experimental conditions used correlate well to those deduced from physical biochemical studies Seifried et al., accompanying paper).

• Seifried SE, Bjornson KP, von Hippel PH
• Structure and assembly of the Escherichia coli transcription termination factor rho and its interactions with RNA. II. Physical chemical studies.
• J Mol Biol. 1991; 221: 1139-51
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• Transcription termination factor rho from Escherichia coli is comprised of a hexamer of identical protein monomers. Hydrodynamic and light-scattering studies have shown the fully assembled rho to be a doughnut-shaped structure. Semi-denaturing gels, protein crosslinking, and spectroscopic studies, as well as other functional and binding determinations have established that the rho hexamer displays D3 symmetry (i.e. it exists as a trimer of dimers). In the accompanying paper we visualize rho directly in the absence of cofactor and show that binding of RNA it into the hexameric form. In this paper we examine the pathway and association constants involved in rho oligomer assembly. Sedimentation and fluorescence-detected size exclusion chromatography are used to demonstrate three steps in the assembly process. These steps can be differentiated by subunit association affinity and kinetic properties. The kinetics of the monomer-dimer equilibrium are fast and an apparent association constant of 1.3 x 10(6) M-1 is measured for this process. In contrast, the dimer-tetramer and tetramer-hexamer association processes appear to be slower (of the order of seconds) and to involve association constants that are smaller than that of the monomer-dimer reaction. This behaviour is consistent with a hexamer of D3 symmetry. Such a particle displays two kinds of subunit interactions; one associated with an intra-dimer A:A interface and the other with an inter-dimer B:B interface. The closure of the circular hexamer does not appear to contribute additional free energy to the assembly process. Fluorescence and sedimentation studies show the association steps to be sensitive to salt concentration. Consistent with earlier work, we find that assembly to the hexameric state is driven by RNA binding.

• Wilson KS, Vorgias CE, Tanaka I, White SW, Kimura M
• The thermostability of DNA-binding protein HU from bacilli.
• Protein Eng. 1990; 4: 11-22
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• The primary and tertiary structures of DNA-binding protein HU from Bacillus stearothermophilus are already known. The primary structure has been previously determined for HU from the closely related B. globigii and the determinations of the sequences from B. caldolyticus and B. subtilis are described here. These bacteria have optimum growth temperatures of greater than 70 degrees C (B. caldolyticus), 65 degrees C (B. stearothermophilus), 37 degrees C (B. subtilis) and 30 degrees C (B. globigii). In vitro measurements from circular dichroic spectra described here give Tm values reflecting these growth temperatures, of 68, 64, 43 and 41 degrees C respectively. We discuss here the relative thermostability of the four proteins in terms of the amino acid differences between the sequences and the three-dimensional model of the B. stearothermophilus HU. The current model for the interaction of the protein with DNA is only discussed in terms of its relevance with regard to thermostability.

• Dombroski AJ, Platt T
• Structure of rho factor: an RNA-binding domain and a separate region with strong similarity to proven ATP-binding domains.
• Proc Natl Acad Sci U S A. 1988; 85: 2538-42
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• The domain structure of rho protein, a transcription termination factor of Escherichia coli, was analyzed by oligonucleotide site-directed mutagenesis and chemical modification methods. The single cysteine at position 202, previously thought to be essential for rho function, was changed to serine or to glycine with no detectable effects on the protein's hexameric structure, RNA-binding ability, or ATPase, helicase, and transcription termination activities. A 151-residue amino-terminal fragment (N1), generated by hydroxylamine cleavage, and its complementary carboxyl-terminal fragment of 268 amino acids (N2) were extracted from NaDod-SO4/polyacrylamide gels and renatured. The N1 fragment binds poly(C) and mRNA corresponding to the rho-dependent terminator sequence trp t', but not RNA unrecognized by rho; hence, this small renaturable domain retains not only the binding ability but also the specificity of the native protein. Uncleaved rho renatures to regain its RNA-dependent ATPase activity, but neither N1 nor N2 exhibits any detectable ATP hydrolysis. Similarly, the two fragments, isolated separately but renatured together, are unable to hydrolyze ATP. Sequence homology to the alpha subunit of the E. coli F1 membrane ATPase, and to consensus elements of other nucleotide-binding proteins, strongly suggests a structural domain for ATP binding that begins after amino acid 164. The implications of discrete domains for RNA and nucleotide binding are discussed in the context of requirements for specific interactions between RNA-binding and ATP-hydrolysis sites during transcription termination.

• McSwiggen JA, Bear DG, von Hippel PH
• Interactions of Escherichia coli transcription termination factor rho with RNA. I. Binding stoichiometries and free energies.
• J Mol Biol. 1988; 199: 609-22
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• In this paper we examine the binding of Escherichia coli transcription termination factor rho to single-stranded RNA. Random polyribonucleotide copolymers containing low ratios of the fluorescent base 1,N6-ethenoadenosine have been synthesized using polynucleotide phosphorylase. Binding of rho to these polynucleotides elicits a significant increase in fluorescence, thus allowing either the direct monitoring of the titration of these polynucleotides with rho or measurement of the competitive displacement of the protein from these probes with other nucleic acids, even in the presence of biologically significant concentrations of ATP. By these techniques, it is shown that the binding site size (n) of rho protein to polynucleotides is 13(+/- 1) nucleotide residues per rho monomer (or 78(+/- 6) nucleotide residues per rho hexamer). Binding constants (K) and co-operativity parameters (omega) for the binding of rho to these polynucleotides have been measured as a function of nucleotide composition and of salt concentration. The results show that the affinity of rho for cytosine residues is quite strong and salt concentration independent, whilst binding to uridine residues is somewhat weaker and very salt concentration dependent. Poly(rC) and poly(dC) bind to rho competitively and with equal affinity and site size, although poly(rC) is the strongest cofactor for activating rho-dependent ATPase and poly(dC) has no ATPase cofactor activity at all. It is also shown that ATP (or ADP or ATP-gamma-S) binding does not change the binding site size of rho on RNA nor decrease its affinity for RNA binding. Circular dichroism measurements of rho binding to phage R17 RNA suggest that the affinity (K omega) of rho for RNA may be increased by ATP. The possible significance of these results for models of rho-dependent transcription termination is discussed in the companion paper.

• Bear DG et al.
• Escherichia coli transcription termination factor rho has a two-domain structure in its activated form.
• Proc Natl Acad Sci U S A. 1985; 82: 1911-5
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• Limited tryptic digestion of Escherichia coli transcription termination factor rho [an RNA-dependent nucleoside triphosphatase (NTPase)] yields predominantly two fragments (f1 and f2) when the protein is bound to both poly(C) and ATP. The apparent molecular masses of the two fragments are 31 kDa for f1 and 15 kDa for f2, adding up to the molecular mass of the intact rho polypeptide chain (46 kDa). Sequence analysis of the amino termini demonstrates that f1 is derived from the amino-terminal portion of rho and that the trypsin cleavage that defines f2 occurs at lysine-283. These results suggest that, in the liganded (activated) form, the native rho protein monomer is organized into two distinct structural domains that are separable by a single proteolytic cleavage. The f1 fragment, purified from NaDodSO4/polyacrylamide gels and renatured, binds poly(C) but the f2 fragment does not; neither regains any ATPase activity. ATP- and polynucleotide-dependent changes in the rate of proteolysis and in the character of the fragments produced suggest that rho undergoes a series of conformational transitions as a consequence of RNA binding, NTP binding and NTP hydrolysis. The rate of loss of rho ATPase activity and of intact rho monomers is slower in the presence of adenosine 5'-[gamma-thio]triphosphate than in the presence of either ATP or ADP, indicating that the hydrolysis of ATP may result in different conformational effects than does the binding of this ligand. These findings are discussed within the context of recent models of rho-dependent transcription termination.

• Engel D, Richardson JP
• Conformational alterations of transcription termination protein rho induced by ATP and by RNA.
• Nucleic Acids Res. 1984; 12: 7389-400
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• Transcription termination protein rho from Escherichia coli possesses an RNA-dependent ATP hydrolysis activity necessary for expression of its termination function. We have used the rate of trypsin-mediated inactivation of ATPase activity as a conformational probe to test for ligand binding-induced conformational changes in the rho polypeptide. When present in molar excess over rho polypeptide, trypsin inactivates rho ATPase by a first order process that correlates well with the loss of intact rho polypeptide. When rho protein binds poly(C) or poly(dC), its susceptible bonds become more accessible to trypsin action. On the other hand, when rho binds either ATP or ADP those bonds become less accessible. These results suggest that rho protein assumes an altered conformation when an RNA cofactor is bound and that is assumes a distinctly different conformation when a nucleotide substrate or product is bound. A special change in the accessibility of trypsin-susceptible bonds is also detected when rho in its complex with poly(C) is catalyzing the hydrolysis of ATP.

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