SMART MODE:

NORMAL GENOMIC

• Martinez VP, Deho G, Simons RW, Garcia-Mena J
• Ribonuclease PH interacts with an acidic ribonuclease E site through a basic 80-amino acid domain.
• FEMS Microbiol Lett. 2014; 355: 51-60
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• In this work, we characterize the domains for the in vivo interaction between ribonuclease E (RNase E) and ribonuclease PH (RNase PH). We initially explored the interaction using pull-down assays with full wild-type proteins expressed from a chromosomal monocopy gene. Once the interaction was confirmed, we narrowed down the sites of interaction in each enzyme to an acidic 16-amino acid region in the carboxy-terminal domain of RNase E and a basic 80-amino acid region in RNase PH including an alpha3 helix. Our results suggest two novel functional domains of interaction between ribonucleases.

• Matos RG, Barria C, Moreira RN, Barahona S, Domingues S, Arraiano CM
• The importance of proteins of the RNase II/RNB-family in pathogenic bacteria.
• Front Cell Infect Microbiol. 2014; 4: 68-68

• Matos RG, Lopez-Vinas E, Gomez-Puertas P, Arraiano CM
• The only exoribonuclease present in Haloferax volcanii has an unique response to temperature changes.
• Biochim Biophys Acta. 2012; 1820: 1543-52
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• BACKGROUND: Little is known regarding mRNA degradation mechanisms in archaea. In some of these single-cell organisms the existence of a complex of exoribonucleases called the exosome has been demonstrated. However, in halophilic archaea the RNase R homologue is essential since it is the only enzyme described with exoribonucleolytic activity. METHODS: In this work we have characterized the mechanism of action of Haloferax volcanii RNase R and its implications for the RNA degradation process. We have determined the salt, pH and divalent ion preference, and set the best conditions for the activity assays. Furthermore, we have determined the activity of the protein at different temperatures using different substrates. The dissociation constants were also calculated by Surface Plasmon Resonance. Finally, we have built a model and compared it with the Escherichia coli counterparts. RESULTS: The results obtained showed that at 37 degrees C, in spite of being named RNase R, this protein behaves like an RNase II protein, halting when it reaches secondary structures, and releasing a 4 nt end-product. However, at 42 degrees C, the optimum temperature of growth, this protein is able to degrade secondary structures, acting like RNase R. GENERAL SIGNIFICANCE: This discovery has a great impact for RNA degradation, since this is the first case reported where a single enzyme has two different exoribonucleolytic activities according to the temperature. Furthermore, the results obtained are very important to help to decipher the RNA degradation mechanisms in H. volcanii, since RNase R is the only exoribonuclease involved in this process.

• Mamolen M, Smith A, Andrulis ED
• Drosophila melanogaster Dis3 N-terminal domains are required for ribonuclease activities, nuclear localization and exosome interactions.
• Nucleic Acids Res. 2010; 38: 5507-17
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• Eukaryotic cells use numerous pathways to regulate RNA production, localization and stability. Several of these pathways are controlled by ribonucleases. The essential ribonuclease, Dis3, plays important roles in distinct RNA metabolic pathways. Despite much progress in understanding general characteristics of the Dis3 enzyme in vitro and in vivo, much less is known about the contributions of Dis3 domains to its activities, subcellular localization and protein-protein interactions. To address these gaps, we constructed a set of Drosophila melanogaster Dis3 (dDis3) mutants and assessed their enzymatic activity in vitro and their localizations and interactions in S2 tissue culture cells. We show that the dDis3 N-terminus is sufficient for endoribonuclease activity in vitro and that proper N-terminal domain structure is critical for activity of the full-length polypeptide. We find that the dDis3 N-terminus also contributes to its subcellular distribution, and is necessary and sufficient for interactions with core exosome proteins. Finally, dDis3 interaction with dRrp6 and dImportin-alpha3 is independent of core interactions and occurs though two different regions. Taken together, our data suggest that the dDis3 N-terminus is a dynamic and complex hub for RNA metabolism and exosome interactions.

• Vincent HA, Deutscher MP
• Insights into how RNase R degrades structured RNA: analysis of the nuclease domain.
• J Mol Biol. 2009; 387: 570-83
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• RNase R readily degrades highly structured RNA, whereas its paralogue, RNase II, is unable to do so. Furthermore, the nuclease domain of RNase R, devoid of all canonical RNA-binding domains, is sufficient for this activity. RNase R also binds RNA more tightly within its catalytic channel than does RNase II, which is thought to be important for its unique catalytic properties. To investigate this idea further, certain residues within the nuclease domain channel of RNase R were changed to those found in RNase II. Among the many examined, we identified one amino acid residue, R572, that has a significant role in the properties of RNase R. Conversion of this residue to lysine, as found in RNase II, results in weaker substrate binding within the nuclease domain channel, longer limit products, increased activity against a variety of substrates and a faster substrate on-rate. Most importantly, the mutant encounters difficulty in degrading structured RNA, pausing within a double-stranded region. Additional studies show that degradation of structured substrates is dependent upon temperature, suggesting a role for thermal breathing in the mechanism of action of RNase R. On the basis of these data, we propose a model in which tight binding within the nuclease domain allows RNase R to capitalize on the natural thermal breathing of an RNA duplex to degrade structured RNAs.

• McGuire AT, Keates RA, Cook S, Mangroo D
• Structural modeling identified the tRNA-binding domain of Utp8p, an essential nucleolar component of the nuclear tRNA export machinery of Saccharomyces cerevisiae.
• Biochem Cell Biol. 2009; 87: 431-43
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• Utp8p is an essential 80 kDa intranuclear tRNA chaperone that transports tRNAs from the nucleolus to the nuclear tRNA export receptors in Saccharomyces cerevisiae. To help understand the mechanism of Utp8p function, predictive tools were used to derive a partial model of the tertiary structure of Utp8p. Secondary structure prediction, supported by circular dichroism measurements, indicated that Utp8p is divided into 2 domains: the N-terminal beta sheet and the C-terminal alpha helical domain. Tertiary structure prediction was more challenging, because the amino acid sequence of Utp8p is not directly homologous to any known protein structure. The tertiary structures predicted by threading and fold recognition had generally modest scores, but for the C-terminal domain, threading and fold recognition consistently pointed to an alpha-alpha superhelix. Because of the sequence diversity of this fold type, no single structural template was an ideal fit to the Utp8p sequence. Instead, a composite template was constructed from 3 different alpha-alpha superhelix structures that gave the best matches to different portions of the C-terminal domain sequence. In the resulting model, the most conserved sequences grouped in a tight cluster of positive charges on a protein that is otherwise predominantly negative, suggesting that the positive-charge cleft may be the tRNA-binding site. Mutations of conserved positive residues in the proposed binding site resulted in a reduction in the affinity of Utp8p for tRNA both in vivo and in vitro. Models were also derived for the 10 fungal homologues of Utp8p, and the localization of the positive charges on the conserved surface was found in all cases. Taken together, these data suggest that the positive-charge cleft of the C-terminal domain of Utp8p is involved in tRNA-binding.

• Dasgupta S, Saha R, Dey C, Banerjee R, Roy S, Basu G
• The role of the catalytic domain of E. coli GluRS in tRNAGln discrimination.
• FEBS Lett. 2009; 583: 2114-20
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• Discrimination of tRNA(Gln) is an integral function of several bacterial glutamyl-tRNA synthetases (GluRS). The origin of the discrimination is thought to arise from unfavorable interactions between tRNA(Gln) and the anticodon-binding domain of GluRS. From experiments on an anticodon-binding domain truncated Escherichia coli (E. coli) GluRS (catalytic domain) and a chimeric protein, constructed from the catalytic domain of E. coli GluRS and the anticodon-binding domain of E. coli glutaminyl-tRNA synthetase (GlnRS), we show that both proteins discriminate against E. coli tRNA(Gln). Our results demonstrate that in addition to the anticodon-binding domain, tRNA(Gln) discriminatory elements may be present in the catalytic domain in E. coli GluRS as well.

• Das D et al.
• Crystal structure of a novel Sm-like protein of putative cyanophage origin at 2.60 A resolution.
• Proteins. 2009; 75: 296-307
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• ECX21941 represents a very large family (over 600 members) of novel, ocean metagenome-specific proteins identified by clustering of the dataset from the Global Ocean Sampling expedition. The crystal structure of ECX21941 reveals unexpected similarity to Sm/LSm proteins, which are important RNA-binding proteins, despite no detectable sequence similarity. The ECX21941 protein assembles as a homopentamer in solution and in the crystal structure when expressed in Escherichia coli and represents the first pentameric structure for this Sm/LSm family of proteins, although the actual oligomeric form in vivo is currently not known. The genomic neighborhood analysis of ECX21941 and its homologs combined with sequence similarity searches suggest a cyanophage origin for this protein. The specific functions of members of this family are unknown, but our structure analysis of ECX21941 indicates nucleic acid-binding capabilities and suggests a role in RNA and/or DNA processing.

• Gunawardana D, Cheng HC, Gayler KR
• Identification of functional domains in Arabidopsis thaliana mRNA decapping enzyme (AtDcp2).
• Nucleic Acids Res. 2008; 36: 203-16
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• The Arabidopsis thaliana decapping enzyme (AtDcp2) was characterized by bioinformatics analysis and by biochemical studies of the enzyme and mutants produced by recombinant expression. Three functionally significant regions were detected: (i) a highly disordered C-terminal region with a putative PSD-95, Discs-large, ZO-1 (PDZ) domain-binding motif, (ii) a conserved Nudix box constituting the putative active site and (iii) a putative RNA binding domain consisting of the conserved Box B and a preceding loop region. Mutation of the putative PDZ domain-binding motif improved the stability of recombinant AtDcp2 and secondary mutants expressed in Escherichia coli. Such recombinant AtDcp2 specifically hydrolysed capped mRNA to produce 7-methyl GDP and decapped RNA. AtDcp2 activity was Mn(2+)- or Mg(2+)-dependent and was inhibited by the product 7-methyl GDP. Mutation of the conserved glutamate-154 and glutamate-158 in the Nudix box reduced AtDcp2 activity up to 400-fold and showed that AtDcp2 employs the catalytic mechanism conserved amongst Nudix hydrolases. Unlike many Nudix hydrolases, AtDcp2 is refractory to inhibition by fluoride ions. Decapping was dependent on binding to the mRNA moiety rather than to the 7-methyl diguanosine triphosphate cap of the substrate. Mutational analysis of the putative RNA-binding domain confirmed the functional significance of an 11-residue loop region and the conserved Box B.

• Keren I, Klipcan L, Bezawork-Geleta A, Kolton M, Shaya F, Ostersetzer-Biran O
• Characterization of the molecular basis of group II intron RNA recognition by CRS1-CRM domains.
• J Biol Chem. 2008; 283: 23333-42
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• CRM (chloroplast RNA splicing and ribosome maturation) is a recently recognized RNA-binding domain of ancient origin that has been retained in eukaryotic genomes only within the plant lineage. Whereas in bacteria CRM domains exist as single domain proteins involved in ribosome maturation, in plants they are found in a family of proteins that contain between one and four repeats. Several members of this family with multiple CRM domains have been shown to be required for the splicing of specific plastidic group II introns. Detailed biochemical analysis of one of these factors in maize, CRS1, demonstrated its high affinity and specific binding to the single group II intron whose splicing it facilitates, the plastid-encoded atpF intron RNA. Through its association with two intronic regions, CRS1 guides the folding of atpF intron RNA into its predicted "catalytically active" form. To understand how multiple CRM domains cooperate to achieve high affinity sequence-specific binding to RNA, we analyzed the RNA binding affinity and specificity associated with each individual CRM domain in CRS1; whereas CRM3 bound tightly to the RNA, CRM1 associated specifically with a unique region found within atpF intron domain I. CRM2, which demonstrated only low binding affinity, also seems to form specific interactions with regions localized to domains I, III, and IV. We further show that CRM domains share structural similarities and RNA binding characteristics with the well known RNA recognition motif domain.

• Calhoun SL, Rao AL
• Functional analysis of brome mosaic virus coat protein RNA-interacting domains.
• Arch Virol. 2008; 153: 231-45
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• The coat proteins (CP) of cowpea chlorotic mottle (CCMV) and brome mosaic virus (BMV), two members of the genus Bromovirus, share 70% identity at the amino acid (aa) level and contain four highly conserved regions, identified as putative RNA-interacting domains (RIDs). To assess the contribution of the conserved aa sequence within each RID and the structural features contained therein toward virion assembly and RNA packaging, we engineered a set of fourteen independent mutations (deletions and substitutions) encompassing all four RIDs. The effect of each mutation on viral biology, pathogenesis, and RNA packaging was analyzed in whole-plant infection assays. Among the four RIDs, two mutations engineered into the N-proximal domain (RID I) and two of the four mutations engineered into the C-proximal domain (RID IV) proved to be more debilitating (compared to wild-type) while only selected regions in the central domains (RID II or III) showed a detectable effect. Neutral effects were observed when aa residues that are predicted to affect calcium binding were mutated. To further analyze the importance of N and C terminal interactions leading to virus assembly and RNA packaging, four CP hybrids were constructed by precisely exchanging either the N-terminal 77 or the C-terminal 113/112aa between BMV and CCMV. Despite the fact that the CP composition of the hybrid viruses is distinct from either of the parents, the symptom phenotype in Chenopodium quinoa, migration pattern of CP in Western blots and virion mobility in agarose gels was indistinguishable from the respective parent providing the genetic background. Collectively, the data provide insight for assessing the relative importance of each RID during genome packaging and in molecular processes regulating the overall architecture of the assembled virions.

• Arraiano CM, Barbas A, Amblar M
• Characterizing ribonucleases in vitro examples of synergies between biochemical and structural analysis.
• Methods Enzymol. 2008; 447: 131-60
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• The contribution of RNA degradation to the posttranscriptional control of gene expression confers on it a fundamental role in all biological processes. Ribonucleases (RNases) are essential enzymes that process and degrade RNA and constitute one of the main groups of factors that determine RNA levels in the cells. RNase II is a ubiquitous, highly processive hydrolytic exoribonuclease that plays an important role in RNA metabolism. This ribonuclease can act independently or as a component of the exosome, an essential RNA-degrading multiprotein complex. In this chapter, we explain the general procedures normally used for the characterization of ribonucleases, using as an example a study performed with Escherichia coli RNase II. We present the overexpression and purification of RNase II recombinant enzyme and of a large set of RNase II truncations. We also describe several methods that can be used for biochemically characterizing the exoribonucleolytic activity and studying RNA binding in vitro. Dissociation constants were determined by electrophoretic mobility shift assay (EMSA), surface plasmon resonance (SPR), and filter binding assays using different single- or double-stranded RNA substrates. We discuss the synergies among the biochemical analyses and the structural studies. These methods will be very useful for the study of other ribonucleases.

• Chalissery J, Banerjee S, Bandey I, Sen R
• Transcription termination defective mutants of Rho: role of different functions of Rho in releasing RNA from the elongation complex.
• J Mol Biol. 2007; 371: 855-72
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• The transcription termination factor Rho of Escherichia coli is a RNA binding protein which can translocate along the RNA and unwind the RNA:DNA hybrid using the RNA-dependent ATPase activity. In order to investigate the involvement of each of these functions in releasing RNA from the elongation complex, we have isolated different termination defective mutants of Rho by random mutagenesis, characterized them for their different functions and established the structure-function correlations from the available structural data of Rho. These mutations are located within the two domains; the N-terminal RNA binding domain (G51V, G53V, and Y80C) and in the C-terminal ATP binding domain (Y274D, P279S, P279L, G324D, N340S, I382N) including the two important structural elements, the Q-loop (P279S, P279L) and R-loop (G324D). Termination defects of the mutants in primary RNA binding domain and Q-loop could not be restored under any conditions that we tested and these were also defective for most of the other functions of Rho. The termination defects of the mutants (Y274D, G324D and N340S), which were mainly defective for secondary RNA binding and likely defective for translocase activity, could be restored under relaxed in vitro conditions. We also show that a mutation in a primary RNA binding domain (Y80C) can cause a defect in ATP binding and induce distinct conformational changes in the distal C-terminal domain, and these allosteric effects are not predictable from the crystal structure. We conclude that the interactions in the primary RNA binding domain and in the Q-loop are mandatory for RNA release to occur and propose that the interactions in the primary RNA binding modulate most of the other functions of Rho allosterically. The rate of ATP hydrolysis regulates the processivity of translocation along the RNA and is directly correlated with the efficiency of RNA release. NusG improves the speed of RNA release and is not involved in any other step.

• Schneider C, Anderson JT, Tollervey D
• The exosome subunit Rrp44 plays a direct role in RNA substrate recognition.
• Mol Cell. 2007; 27: 324-31
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• The exosome plays key roles in RNA maturation and surveillance, but it is unclear how target RNAs are identified. We report the functional characterization of the yeast exosome component Rrp44, a member of the RNase II family. Recombinant Rrp44 and the purified TRAMP polyadenylation complex each specifically recognized tRNA(i)(Met) lacking a single m(1)A(58) modification, even in the presence of a large excess of total tRNA. This tRNA is otherwise mature and functional in translation in vivo but is presumably subtly misfolded. Complete degradation of the hypomodified tRNA required both Rrp44 and the poly(A) polymerase activity of TRAMP. The intact exosome lacking only the catalytic activity of Rrp44 failed to degrade tRNA(i)(Met), showing this to be a specific Rrp44 substrate. Recognition of hypomodified tRNA(i)(Met) by Rrp44 is genetically separable from its catalytic activity on other substrates, with the mutations mapping to distinct regions of the protein.

• Tadokoro T, Chon H, Koga Y, Takano K, Kanaya S
• Identification of the gene encoding a type 1 RNase H with an N-terminal double-stranded RNA binding domain from a psychrotrophic bacterium.
• FEBS J. 2007; 274: 3715-27
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• The gene encoding a bacterial type 1 RNase H, termed RBD-RNase HI, was cloned from the psychrotrophic bacterium Shewanella sp. SIB1, overproduced in Escherichia coli, and the recombinant protein was purified and biochemically characterized. SIB1 RBD-RNase HI consists of 262 amino acid residues and shows amino acid sequence identities of 26% to SIB1 RNase HI, 17% to E. coli RNase HI, and 32% to human RNase H1. SIB1 RBD-RNase HI has a double-stranded RNA binding domain (RBD) at the N-terminus, which is commonly present at the N-termini of eukaryotic type 1 RNases H. Gel mobility shift assay indicated that this domain binds to an RNA/DNA hybrid in an isolated form, suggesting that this domain is involved in substrate binding. SIB1 RBD-RNase HI exhibited the enzymatic activity both in vitro and in vivo. Its optimum pH and metal ion requirement were similar to those of SIB1 RNase HI, E. coli RNase HI, and human RNase H1. The specific activity of SIB1 RBD-RNase HI was comparable to that of E. coli RNase HI and was much higher than those of SIB1 RNase HI and human RNase H1. SIB1 RBD-RNase HI showed poor cleavage-site specificity for oligomeric substrates. SIB1 RBD-RNase HI was less stable than E. coli RNase HI but was as stable as human RNase H1. Database searches indicate that several bacteria and archaea contain an RBD-RNase HI. This is the first report on the biochemical characterization of RBD-RNase HI.

• Jenvert RM, Schiavone LH
• The flexible N-terminal domain of ribosomal protein L11 from Escherichia coli is necessary for the activation of stringent factor.
• J Mol Biol. 2007; 365: 764-72
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• The stringent response is activated by the binding of stringent factor to stalled ribosomes that have an unacylated tRNA in the ribosomal aminoacyl-site. Ribosomes lacking ribosomal protein L11 are deficient in stimulating stringent factor. L11 consists of a dynamic N-terminal domain (amino acid residues 1-72) connected to an RNA-binding C-terminal domain (amino acid residues 76-142) by a flexible linker (amino acid residues 73-75). In vivo data show that mutation of proline 22 in the N-terminal domain is important for initiation of the stringent response. Here, six different L11 point and deletion-mutants have been constructed to determine which regions of L11 are necessary for the activation of stringent factor. The different mutants were reconstituted with programmed 70 S(DeltaL11) ribosomes and tested for their ability to stimulate stringent factor in a sensitive in vitro pppGpp synthesis assay. It was found that a single-site mutation at proline 74 in the linker region between the two domains did not affect the stimulatory activity of the reconstituted ribosomes, whereas the single-site mutation at proline 22 reduced the activity of SF to 33% compared to ribosomes reconstituted with wild-type L11. Removal of the entire linker between the N and C-terminal domains or removal of the entire proline-rich helix beginning at proline 22 in L11 resulted in an L11 protein, which was unable to stimulate stringent factor in the ribosome-dependent assay. Surprisingly, the N-terminal domain of L11 on its own activated stringent factor in a ribosome-dependent manner without restoring the L11 footprint in 23 S rRNA in the 50 S subunit. This suggests that the N-terminal domain can activate stringent factor in trans. It is also shown that this activation is dependent on unacylated tRNA.

• Sunita S, Zhenxing H, Swaathi J, Cygler M, Matte A, Sivaraman J
• Domain organization and crystal structure of the catalytic domain of E.coli RluF, a pseudouridine synthase that acts on 23S rRNA.
• J Mol Biol. 2006; 359: 998-1009
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• Pseudouridine synthases catalyze the isomerization of uridine to pseudouridine (Psi) in rRNA and tRNA. The pseudouridine synthase RluF from Escherichia coli (E.C. 4.2.1.70) modifies U2604 in 23S rRNA, and belongs to a large family of pseudouridine synthases present in all kingdoms of life. Here we report the domain architecture and crystal structure of the catalytic domain of E.coli RluF at 2.6A resolution. Limited proteolysis, mass spectrometry and N-terminal sequencing indicate that RluF has a distinct domain architecture, with the catalytic domain flanked at the N and C termini by additional domains connected to it by flexible linkers. The structure of the catalytic domain of RluF is similar to those of RsuA and TruB. RluF is a member of the RsuA sequence family of Psi-synthases, along with RluB and RluE. Structural comparison of RluF with its closest structural homologues, RsuA and TruB, suggests possible functional roles for the N-terminal and C-terminal domains of RluF.

• Anderson JR, Mukherjee D, Muthukumaraswamy K, Moraes KC, Wilusz CJ, Wilusz J
• Sequence-specific RNA binding mediated by the RNase PH domain of components of the exosome.
• RNA. 2006; 12: 1810-6
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• We have previously demonstrated that PM-Scl-75, a component of the human exosome complex involved in RNA maturation and mRNA decay, can specifically interact with RNAs containing an AU-rich instability element. Through the analysis of a series of deletion mutants, we have now shown that a 266 amino acid fragment representing the RNase PH domain is responsible for the sequence-specific binding to AU-rich elements. Furthermore, we found that the RNase PH domains from two other exosomal components, OIP2 and RRP41, as well as from Escherichia coli polynucleotide phosphorylase, are all capable of specifically interacting with RNAs containing an AU-rich element with similar affinities. Finally, we demonstrate that the interaction of the RNase PH domain of PM-Scl-75 is readily competed by poly(U), but only inefficiently using other homopolymeric RNAs. These data demonstrate that RNase PH domains in general have an affinity for U- and AU-rich sequences, and broaden the potential role in RNA biology of proteins containing these domains.

• Gamberi C, Johnstone O, Lasko P
• Drosophila RNA binding proteins.
• Int Rev Cytol. 2006; 248: 43-139
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• RNA binding proteins are fundamental mediators of gene expression. The use of the model organism Drosophila has helped to elucidate both tissue-specific and ubiquitous functions of RNA binding proteins. These proteins mediate all aspects of the mRNA lifespan including splicing, nucleocytoplasmic transport, localization, stability, translation, and degradation. Most RNA binding proteins fall into several major groups, based on their RNA binding domains. As well, experimental data have revealed several proteins that can bind RNA but lack canonical RNA binding motifs, suggesting the presence of as yet uncharacterized RNA binding domains. Here, we present the major classes of Drosophila RNA binding proteins with special focus on those with functional information.

• Richards J, Mehta P, Karzai AW
• RNase R degrades non-stop mRNAs selectively in an SmpB-tmRNA-dependent manner.
• Mol Microbiol. 2006; 62: 1700-12
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• The SmpB-tmRNA-mediated trans-translation system has two well-established activities: rescuing ribosomes stalled on aberrant mRNAs and marking the associated protein fragments for proteolysis. Although the causative non-stop mRNAs are known to be degraded, little is known about the enabling mechanism or the RNases involved in their disposal. We report that Escherichia coli has an enabling mechanism that requires RNase R activity and is dependent on the presence of SmpB protein and tmRNA, suggesting a requirement for active transtranslation in facilitating RNase R engagement and promoting non-stop mRNA decay. Interestingly, this selective transcript degradation by RNase R targets aberrant (non-stop and multiple-rare-codon containing) mRNAs and does not affect the decay of related messages containing in-frame stop codons. Most surprisingly, RNase II and PNPase do not play a significant role in tmRNA-facilitated disposal of aberrant mRNAs. These findings demonstrate that RNase R is a crucial component of the trans-translation-mediated non-stop mRNA decay process, thus providing a requisite activity well suited to complement the ribosome rescue and protein tagging functions of this unique quality control system.

• Sutherland LC, Rintala-Maki ND, White RD, Morin CD
• RNA binding motif (RBM) proteins: a novel family of apoptosis modulators?
• J Cell Biochem. 2005; 94: 5-24
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• RBM5 is a known modulator of apoptosis, an RNA binding protein, and a putative tumor suppressor. Originally identified as LUCA-15, and subsequently as H37, it was designated "RBM" (for RNA Binding Motif) due to the presence of two RRM (RNA Recognition Motif) domains within the protein coding sequence. Recently, a number of proteins have been attributed with this same RBM designation, based on the presence of one or more RRM consensus sequences. One such protein, RBM3, was also recently found to have apoptotic modulatory capabilities. The high sequence homology at the amino acid level between RBM5, RBM6, and particularly, RBM10 suggests that they, too, may play an important role in regulating apoptosis. It is the intent of this article to ammalgamate the data on the ten originally identified RBM proteins in order to question the existence of a novel family of RNA binding apoptosis regulators.

• Gan J, Tropea JE, Austin BP, Court DL, Waugh DS, Ji X
• Intermediate states of ribonuclease III in complex with double-stranded RNA.
• Structure. 2005; 13: 1435-42
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• Bacterial ribonuclease III (RNase III) can affect RNA structure and gene expression in either of two ways: as a processing enzyme that cleaves double-stranded (ds) RNA, or as a binding protein that binds but does not cleave dsRNA. We previously proposed a model of the catalytic complex of RNase III with dsRNA based on three crystal structures, including the endonuclease domain of RNase III with and without bound metal ions and a dsRNA binding protein complexed with dsRNA. We also reported a noncatalytic assembly observed in the crystal structure of an RNase III mutant, which binds but does not cleave dsRNA, complexed with dsRNA. We hypothesize that the RNase III*dsRNA complex can exist in two functional forms, a catalytic complex and a noncatalytic assembly, and that in between the two forms there may be intermediate states. Here, we present four crystal structures of RNase III complexed with dsRNA, representing possible intermediates.

• Athanasiadis A, Placido D, Maas S, Brown BA 2nd, Lowenhaupt K, Rich A
• The crystal structure of the Zbeta domain of the RNA-editing enzyme ADAR1 reveals distinct conserved surfaces among Z-domains.
• J Mol Biol. 2005; 351: 496-507
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• The Zalpha domains represent a growing subfamily of the winged helix-turn-helix (HTH) domain family whose members share a remarkable ability to bind specifically to Z-DNA and/or Z-RNA. They have been found exclusively in proteins involved in interferon response and, while their importance in determining pox viral pathogenicity has been demonstrated, their actual target and biological role remain obscure. Cellular proteins containing Zalpha domains bear a second homologous domain termed Zbeta, which appears to lack the ability to bind left-handed nucleic acids. Here, we present the crystal structure of the Zbeta domain from the human double-stranded RNA adenosine deaminase ADAR1 at 0.97 A, determined by single isomorphous replacement including anomalous scattering. Zbeta maintains a winged-HTH fold with the addition of a C-terminal helix. Mapping of the Zbeta conservation profile on the Zbeta surface reveals a new conserved surface formed partly by the terminal helix 4, involved in metal binding and dimerization and absent from Zalpha domains. Our results show how two domains similar in fold may have evolved into different functional entities even in the context of the same protein.

• Yang L, Wesolowski D, Li Y, Altman S
• Analysis of putative RNase P RNA from orthopoxviruses.
• J Mol Biol. 2005; 354: 529-35
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• A putative RNase P RNA gene in camelpox virus, one of the orthopoxviruses, was cloned and transcribed in vitro. No RNase P activity could be detected in vitro from camelpox virus RNase P RNA alone, or by addition of the Escherichia coli RNase P protein subunit to reaction mixtures. Camelpox virus RNase P RNA reconstituted in vitro with camel or HeLa cell extracts, which were pre-treated with micrococcal nuclease to degrade the endogenous RNase P RNA, showed no RNase P activity. Vaccinia virus, another orthopoxvirus, showed no RNase P activity in vaccinia-infected HeLa cells, even though transcription of the vaccinia RNase P RNA could be identified in the cells by both Northern blot and RNase protection assay. Camelpox virus RNase P RNA inhibited an endogenous HeLa RNase P activity by 20% in our assays. The 5S RNA showed no significant inhibition in this assay.

• Bermudez-Cruz RM, Fernandez-Ramirez F, Kameyama-Kawabe L, Montanez C
• Conserved domains in polynucleotide phosphorylase among eubacteria.
• Biochimie. 2005; 87: 737-45
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• Polynucleotide phosphorylase (PNPase) is a polynucleotide nucleotidyl transferase (E. C. 2.7.7.8) that is involved in mRNA degradation in prokaryotes. PNPase structure analysis has been performed in Streptomyces antibioticus; this revealed the presence of five domains: two ribonuclease PH (RPH)-like (pnp1 and pnp2), one alpha helical, one KH, and one S1 domains. The trimeric nature of this enzyme was also confirmed. In this work, we have investigated conserved domains or subdomains in bacterial PNPases (55), for this structure-based sequence homology analysis between predicted amino acid sequences from bacterial PNPases and that of S. antibioticus was performed. Our findings indicate that while pnp2 (% similarity average S = 84/% identity average I = 22), KH (S = 74.3%/I = 5.3%), S1 (S = 71.3%/I = 1.2%); and pnp1 (S = 52.8%/I = 0.3%) domain; structure and sequence are well conserved among different bacteria, alpha helical domain (S = 39.5%/I = 0) although conservation of the structure is somewhat maintained, the sequence is not conserved at all. Implications of such findings in PNPase activity will be discussed.

• Spencer AC, Spremulli LL
• The interaction of mitochondrial translational initiation factor 2 with the small ribosomal subunit.
• Biochim Biophys Acta. 2005; 1750: 69-81
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• Bovine mitochondrial translational initiation factor 2 (IF-2(mt)) is organized into four domains, an N-terminal domain, a central G-domain and two C-terminal domains. These domains correspond to domains III-VI in the six-domain model of Escherichia coli IF-2. Variants in IF-2(mt) were prepared and tested for their abilities to bind the small (28S) subunit of the mitochondrial ribosome. The binding of wild-type IF-2(mt) was strong (K(d) approximately 10-20 nM) and was not affected by fMet-tRNA. Deletion of the N-terminal domain substantially reduced the binding of IF-2(mt) to 28S subunits. However, the addition of fMet-tRNA stimulated the binding of this variant at least 2-fold demonstrating that contacts between fMet-tRNA and IF-2(mt) can stabilize the binding of this factor to 28S subunits. No binding was observed for IF-2(mt) variants lacking the G-domain which probably plays a critical role in organizing the structure of IF-2(mt). IF-2(mt) contains a 37-amino acid insertion region between domains V and VI that is not found in the prokaryotic factors. Mutations in this region caused a significant reduction in the ability of the factor to promote initiation complex formation and to bind 28S subunits.

• Nakaminami K et al.
• Heat stable ssDNA/RNA-binding activity of a wheat cold shock domain protein.
• FEBS Lett. 2005; 579: 4887-91
• Display abstract

• The cold-induced wheat WCSP1 protein belongs to the cold shock domain (CSD) protein family. In prokaryotes and eukaryotes, the CSD functions as a nucleic acid-binding domain. Here, we demonstrated that purified recombinant WCSP1 is boiling soluble and binds ss/dsDNA and mRNA. Furthermore, boiled-WCSP1 retained its characteristic nucleic acid-binding activity. A WCSP1 deletion mutant, containing only a CSD, lost ssDNA/RNA-binding activity; while a mutant containing the CSD and the first glycine-rich region (GR) displayed the activity. These data indicated that the first GR of WCSP1 is necessary for the binding activity but is not for the heat stability of the protein.

• de Waal PJ, Huismans H
• Characterization of the nucleic acid binding activity of inner core protein VP6 of African horse sickness virus.
• Arch Virol. 2005; 150: 2037-50
• Display abstract

• Minor structural protein VP6 is the putative helicase of African horse sickness virus (AHSV), of the genus Orbivirus in the Reoviridae family. We investigated how the protein interacts with double-stranded (ds) RNA and other nucleic acids. Binding was assayed using an electrophoretic migration retardation assay and a nucleic acid overlay protein blot assay. VP6 bound double and single stranded RNA and DNA in a NaCl concentration sensitive reaction. Of six truncated VP6 peptides investigated, two partially overlapping peptides were found to bind dsRNA at pH 7.0, while other peptides with the same overlap did not. The distinction between the peptides appeared to be the pI which ranged from more than 8.0 to just above 6.0. Changing the pH of the binding buffer modified the binding activity. Regardless of assay conditions, only peptides with a specific region of amino acids in common, showed evidence of binding activity. No sequence homology was identified with other binding domains, however, the presence of charged amino acids are assumed to be important for binding activity. The results suggested dsRNA binding in the blot assay was strongly affected by the net charge on the peptide.

• Leulliot N et al.
• A new alpha-helical extension promotes RNA binding by the dsRBD of Rnt1p RNAse III.
• EMBO J. 2004; 23: 2468-77
• Display abstract

• Rnt1 endoribonuclease, the yeast homolog of RNAse III, plays an important role in the maturation of a diverse set of RNAs. The enzymatic activity requires a conserved catalytic domain, while RNA binding requires the double-stranded RNA-binding domain (dsRBD) at the C-terminus of the protein. While bacterial RNAse III enzymes cleave double-stranded RNA, Rnt1p specifically cleaves RNAs that possess short irregular stem-loops containing 12-14 base pairs interrupted by internal loops and bulges and capped by conserved AGNN tetraloops. Consistent with this substrate specificity, the isolated Rnt1p dsRBD and the 30-40 amino acids that follow bind to AGNN-containing stem-loops preferentially in vitro. In order to understand how Rnt1p recognizes its cognate processing sites, we have defined its minimal RNA-binding domain and determined its structure by solution NMR spectroscopy and X-ray crystallography. We observe a new carboxy-terminal helix following a canonical dsRBD structure. Removal of this helix reduces binding to Rnt1p substrates. The results suggest that this helix allows the Rnt1p dsRBD to bind to short RNA stem-loops by modulating the conformation of helix alpha1, a key RNA-recognition element of the dsRBD.

• Callaghan AJ et al.
• Studies of the RNA degradosome-organizing domain of the Escherichia coli ribonuclease RNase E.
• J Mol Biol. 2004; 340: 965-79
• Display abstract

• The hydrolytic endoribonuclease RNase E, which is widely distributed in bacteria and plants, plays key roles in mRNA degradation and RNA processing in Escherichia coli. The enzymatic activity of RNase E is contained within the conserved amino-terminal half of the 118 kDa protein, and the carboxy-terminal half organizes the RNA degradosome, a multi-enzyme complex that degrades mRNA co-operatively and processes ribosomal and other RNA. The study described herein demonstrates that the carboxy-terminal domain of RNase E has little structure under native conditions and is unlikely to be extensively folded within the degradosome. However, three isolated segments of 10-40 residues, and a larger fourth segment of 80 residues, are predicted to be regions of increased structural propensity. The larger of these segments appears to be a protein-RNA interaction site while the other segments possibly correspond to sites of self-recognition and interaction with the other degradosome proteins. The carboxy-terminal domain of RNase E may thus act as a flexible tether of the degradosome components. The implications of these and other observations for the organization of the RNA degradosome are discussed.

• Chen X, Stitt BL
• The binding of C10 oligomers to Escherichia coli transcription termination factor Rho.
• J Biol Chem. 2004; 279: 16301-10
• Display abstract

• The binding of C10 RNA oligomers to wild type and mutant Escherichia coli transcription termination factor Rho provides a model for the enzyme-RNA interactions that lead to transcription termination. One surprising finding is that wild type Rho binds between five and six C10 oligomers per hexamer with KD = 0.3 microm, and five to six additional C10 molecules with KD = 7 microm. Previously, approximately half this number of oligomer-binding sites was reported (Wang, Y., and von Hippel, P. H. (1993) J. Biol. Chem. 268, 13947-13955); however, the E155K mutant form of Rho, thought at the time to be wild type, was used in that work. The present results with E155K Rho agree with the earlier work. C10 binding with mutant forms of Rho that are altered in RNA interactions, bearing amino acid changes F62S, G99V, F232C, T286A, or K352E, indicate that the higher affinity binding sites constitute what has been termed the primary RNA site, and the lower affinity sites constitute the secondary sites. The binding data together with the crystal structures for wild type Rho (Skordalakes, E., and Berger, J. M. (2003) Cell 114, 135-146) support structurally distinct locations on Rho for the two classes of C10-binding sites. The results are consistent with participation of residues 33 A apart in secondary site RNA interactions. The data further indicate that not all RNA sites on Rho must be filled for full ATPase and transcription termination activity, and suggest a model in which RNA binding to the higher affinity sites leads to a protein conformation change that exposes the previously hidden lower affinity sites.

• Sun W, Li G, Nicholson AW
• Mutational analysis of the nuclease domain of Escherichia coli ribonuclease III. Identification of conserved acidic residues that are important for catalytic function in vitro.
• Biochemistry. 2004; 43: 13054-62
• Display abstract

• The ribonuclease III superfamily represents a structurally distinct group of double-strand-specific endonucleases with essential roles in RNA maturation, RNA decay, and gene silencing. Bacterial RNase III orthologs exhibit the simplest structures, with an N-terminal nuclease domain and a C-terminal double-stranded RNA-binding domain (dsRBD), and are active as homodimers. The nuclease domain contains conserved acidic amino acids, which in Escherichia coli RNase III are E38, E41, D45, E65, E100, D114, and E117. On the basis of a previously reported crystal structure of the nuclease domain of Aquifex aeolicus RNase III, the E41, D114, and E117 side chains of E. coli RNase III are expected to be coordinated to a divalent metal ion (Mg(2+) or Mn(2+)). It is shown here that the RNase III[E41A] and RNase III[D114A] mutants exhibit catalytic activities in vitro in 10 mM Mg(2+) buffer that are comparable to that of the wild-type enzyme. However, at 1 mM Mg(2+), the activities are significantly lower, which suggests a weakened affinity for metal. While RNase III[E41A] and RNase III[D114A] have K(Mg) values that are approximately 2.8-fold larger than the K(Mg) of RNase III (0.46 mM), the RNase III[E41A/D114A] double mutant has a K(Mg) of 39 mM, suggesting a redundant function for the two side chains. RNase III[E38A], RNase III[E65A], and RNase III[E100A] also require higher Mg(2+) concentrations for optimal activity, with RNase III[E100A] exhibiting the largest K(Mg). RNase III[D45A], RNase III[D45E], and RNase III[D45N] exhibit negligible activities, regardless of the Mg(2+) concentration, indicating a stringent functional requirement for an aspartate side chain. RNase III[D45E] activity is partially rescued by Mn(2+). The potential functions of the conserved acidic residues are discussed in the context of the crystallographic data and proposed catalytic mechanisms.

• Callaghan AJ et al.
• Quaternary structure and catalytic activity of the Escherichia coli ribonuclease E amino-terminal catalytic domain.
• Biochemistry. 2003; 42: 13848-55
• Display abstract

• RNase E is an essential endoribonuclease that plays a central role in the processing and degradation of RNA in Escherichia coli and other bacteria. Most endoribonucleases have been shown to act distributively; however, Feng et al. [(2002) Proc. Natl. Acad. Sci. U.S.A. 99, 14746-14751] have recently found that RNase E acts via a scanning mechanism. A structural explanation for the processivity of RNase E is provided here, with our finding that the conserved catalytic domain of E. coli RNase E forms a homotetramer. Nondissociating nanoflow-electrospray mass spectrometry suggests that the tetramer binds up to four molecules of a specific substrate RNA analogue. The tetrameric assembly of the N-terminal domain of RNase E is consistent with crystallographic analyses, which indicate that the tetramer possesses approximate D(2) dihedral symmetry. Using X-ray solution scattering data and symmetry restraints, a solution shape is calculated for the tetramer. This shape, together with limited proteolysis data, suggests that the S1-RNA binding domains of RNase E lie on the periphery of the tetramer. These observations have implications for the structure and function of the RNase E/RNase G ribonuclease family and for the assembly of the E. coli RNA degradosome, in which RNase E is the central component.

• Doyle M, Jantsch MF
• Distinct in vivo roles for double-stranded RNA-binding domains of the Xenopus RNA-editing enzyme ADAR1 in chromosomal targeting.
• J Cell Biol. 2003; 161: 309-19
• Display abstract

• The RNA-editing enzyme adenosine deaminase that acts on RNA (ADAR1) deaminates adenosines to inosines in double-stranded RNA substrates. Currently, it is not clear how the enzyme targets and discriminates different substrates in vivo. However, it has been shown that the deaminase domain plays an important role in distinguishing various adenosines within a given substrate RNA in vitro. Previously, we could show that Xenopus ADAR1 is associated with nascent transcripts on transcriptionally active lampbrush chromosomes, indicating that initial substrate binding and possibly editing itself occurs cotranscriptionally. Here, we demonstrate that chromosomal association depends solely on the three double-stranded RNA-binding domains (dsRBDs) found in the central part of ADAR1, but not on the Z-DNA-binding domain in the NH2 terminus nor the catalytic deaminase domain in the COOH terminus of the protein. Most importantly, we show that individual dsRBDs are capable of recognizing different chromosomal sites in an apparently specific manner. Thus, our results not only prove the requirement of dsRBDs for chromosomal targeting, but also show that individual dsRBDs have distinct in vivo localization capabilities that may be important for initial substrate recognition and subsequent editing specificity.

• Kanai A, Oida H, Matsuura N, Doi H
• Expression cloning and characterization of a novel gene that encodes the RNA-binding protein FAU-1 from Pyrococcus furiosus.
• Biochem J. 2003; 372: 253-61
• Display abstract

• We systematically screened a genomic DNA library to identify proteins of the hyperthermophilic archaeon Pyrococcus furiosus using an expression cloning method. One gene product, which we named FAU-1 (P. furiosus AU-binding), demonstrated the strongest binding activity of all the genomic library-derived proteins tested against an AU-rich RNA sequence. The protein was purified to near homogeneity as a 54 kDa single polypeptide, and the gene locus corresponding to this FAU-1 activity was also sequenced. The FAU-1 gene encoded a 472-amino-acid protein that was characterized by highly charged domains consisting of both acidic and basic amino acids. The N-terminal half of the gene had a degree of similarity (25%) with RNase E from Escherichia coli. Five rounds of RNA-binding-site selection and footprinting analysis showed that the FAU-1 protein binds specifically to the AU-rich sequence in a loop region of a possible RNA ligand. Moreover, we demonstrated that the FAU-1 protein acts as an oligomer, and mainly as a trimer. These results showed that the FAU-1 protein is a novel heat-stable protein with an RNA loop-binding characteristic.

• Muroya A, Nakano R, Ohtani N, Haruki M, Morikawa M, Kanaya S
• Importance of an N-terminal extension in ribonuclease HII from Bacillus stearothermophilus for substrate binding.
• J Biosci Bioeng. 2002; 93: 170-5
• Display abstract

• The gene encoding ribonuclease HII from Bacillus stearothermophilus was cloned and expressed in Escherichia coli. The overproduced protein, Bst-RNase HII, was purified and biochemically characterized. Bst-RNase HII, which consists of 259 amino acid residues, showed the highest amino acid sequence identity (50.2%) to Bacillus subtilis RNase HII. Like B. subtilis RNase HII, it exhibited Mn2+-dependent RNase H activity. It was, however, more thermostable than B. subtilis RNase HII. When the Bst-RNase HII amino acid sequence is compared with that of Thermococcus kodakaraensis RNase HII, to which it shows 29.8% identity, 30 residues are observed to be truncated from the C-terminus and there is an extension of 71 residues at the N-terminus. The C-terminal truncation results in the loss of the alpha9 helix, which is rich in basic amino acid residues and is therefore important for substrate binding. A truncated protein, Delta59-Bst-RNase HII, in which most of the N-terminal extension was removed, completely lost its RNase H activity. Surface plasmon resonance analysis indicated that this truncated protein did not bind to the substrate. These results suggest that the N-terminal extension of Bst-RNase HII is important for substrate binding. Because B. subtilis RNase HII has an N-terminal extension of the same length and these extensions contain a region in which basic amino acid residues are clustered, the Bacillus enzymes may represent a novel type of RNase H which possesses a substrate-binding domain at the N-terminus.

• Diwa AA, Jiang X, Schapira M, Belasco JG
• Two distinct regions on the surface of an RNA-binding domain are crucial for RNase E function.
• Mol Microbiol. 2002; 46: 959-69
• Display abstract

• Despite its importance for RNA processing and degradation in Escherichia coli, little is known about the structure of RNase E or its mechanism of action. We have modelled the three-dimensional structure of an essential amino-terminal domain of RNase E on the basis of its sequence homology to the S1 family of RNA-binding domains. Each of the five surface-exposed aromatic residues and most of the 14 basic residues of this RNase E domain were replaced with alanine to determine their importance for RNase E function. All the surface residues essential for cell growth and feedback regulation of RNase E synthesis mapped to one end of the domain. In vitro assays indicate that these essential residues fall into two functionally distinct groups that form discrete clusters on opposite faces of the S1 domain. One group, comprising Phe-57, Phe-67 and Lys-112 [corrected], is of general importance for the ribonuclease activity of RNase E, whereas the other group, comprising Lys-37 and Tyr-60, is entirely dispensable for catalytic activity in vitro. The side-chains of two residues previously identified as sites of temperature-sensitive mutations lie buried directly beneath the surface region defined by Phe-57, Phe-67 and Lys-112 [corrected], which probably enhances RNase E activity by making a crucial contribution to the binding of substrate RNAs. In contrast to the S1 domain, an arginine-rich RNA-binding domain in the carboxyl half of RNase E appears to have a more peripheral role in RNase E function, as it is not required for feedback regulation, cell growth or ribonuclease activity.

• Conrad C, Evguenieva-Hackenberg E, Klug G
• Both N-terminal catalytic and C-terminal RNA binding domain contribute to substrate specificity and cleavage site selection of RNase III.
• FEBS Lett. 2001; 509: 53-8
• Display abstract

• The double-stranded RNA-specific endoribonuclease III (RNase III) of bacteria consists of an N-terminal nuclease domain and a double-stranded RNA binding domain (dsRBD) at the C-terminus. Analysis of two hybrid proteins consisting of the N-terminal half of Escherichia coli RNase III fused to the dsRBD of the Rhodobacter capsulatus enzyme and vice versa reveals that both domains in combination with the particular substrate determine substrate specificity and cleavage site selection. Extension of the spacer between the two domains of the E. coli enzyme from nine to 20 amino acids did not affect cleavage site selection.

• Pompeo F, Bourne Y, van Heijenoort J, Fassy F, Mengin-Lecreulx D
• Dissection of the bifunctional Escherichia coli N-acetylglucosamine-1-phosphate uridyltransferase enzyme into autonomously functional domains and evidence that trimerization is absolutely required for glucosamine-1-phosphate acetyltransferase activity and cell growth.
• J Biol Chem. 2001; 276: 3833-9
• Display abstract

• The bifunctional N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) enzyme catalyzes both the acetylation of glucosamine 1-phosphate and the uridylation of N-acetylglucosamine 1-phosphate, two subsequent steps in the pathway for UDP-N-acetylglucosamine synthesis in bacteria. In our previous work describing its initial characterization in Escherichia coli, we proposed that the 456-amino acid (50.1 kDa) protein might possess separate uridyltransferase (N-terminal) and acetyltransferase (C-terminal) domains. In the present study, we confirm this hypothesis by expression of the two independently folding and functional domains. A fragment containing the N-terminal 331 amino acids (Tr331, 37.1 kDa) has uridyltransferase activity only, with steady-state kinetic parameters similar to the full-length protein. Further deletion of 80 amino acid residues at the C terminus results in a 250-amino acid fragment (28.6 kDa) still exhibiting significant uridyltransferase activity. Conversely, a fragment containing the 233 C-terminal amino acids (24.7 kDa) exhibits acetyltransferase activity exclusively. None of these individual domains could complement a chromosomal glmU mutation, indicating that each of the two activities is essential for cell viability. Analysis of truncated GlmU proteins by gel filtration further localizes regions of the protein involved in its trimeric organization. Interestingly, overproduction of the truncated Tr331 protein in a wild-type strain results in a rapid depletion of endogenous acetyltransferase activity, an arrest of peptidoglycan synthesis and cell lysis. It is shown that the acetyltransferase activity of the full-length protein is abolished once trapped within heterotrimers formed in presence of the truncated protein, suggesting that this enzyme activity absolutely requires a trimeric organization and that the catalytic site involves regions of contact between adjacent monomers. Data are discussed in connection with the recently obtained crystal structure of the truncated Tr331 protein.

• Akiyama T et al.
• Mammalian homologue of E. coli Ras-like GTPase (ERA) is a possible apoptosis regulator with RNA binding activity.
• Genes Cells. 2001; 6: 987-1001
• Display abstract

• BACKGROUND: ERA (Escherichia coli Ras-like protein) is an E. coli GTP binding protein that is essential for proliferation. A DNA database search suggests that homologous sequences with ERA exist in various organisms including human, mouse, Drosophila, Caenorhabditis elegans and Antirrhinum majus. However, the physiological function of eukaryotic ERA-like proteins is not known. RESULTS: We have cloned cDNAs encoding the entire coding region of a human homologue (H-ERA) and a mouse homologue (M-ERA) of ERA. The mammalian homologue of ERA consists of a typical GTPase/GTP-binding domain and a putative K homology (KH) domain, which is known as an RNA binding domain. We performed transfection experiments with wild-type H-ERA or various H-ERA mutants. H-ERA possessing the amino acid substitution mutation into the GTPase domain induced apoptosis of HeLa cells, which was blocked by Bcl-2 expression. Deletion of the C-terminus, which contains a part of the KH domain, alleviated apoptosis by the H-ERA mutant, suggesting the importance of this domain in the function of H-ERA. We have also shown the RNA binding activity of H-ERA by pull-down experiments using RNA homopolymer immobilized on beads or recombinant H-ERA proteins. CONCLUSION: Our data suggest that H-ERA plays an important role in the regulation of apoptotic signalling with its GTPase/GTP binding domain.

• Daviet L, Erard M, Dorin D, Duarte M, Vaquero C, Gatignol A
• Analysis of a binding difference between the two dsRNA-binding domains in TRBP reveals the modular function of a KR-helix motif.
• Eur J Biochem. 2000; 267: 2419-31
• Display abstract

• Double-stranded RNA-binding proteins constitute a large family with conserved domains called dsRBDs. One of these, TRBP, a protein that binds HIV-1 TAR RNA, has two dsRBDs (dsRBD1 and dsRBD2), as indicated by computer sequence homology. However, a 24-amino-acid deletion in dsRBD2 completely abolishes RNA binding, suggesting that only one domain is functional. To analyse further the similarities and differences between these domains, we expressed them independently and measured their RNA-binding affinities. We found that dsRBD2 has a dissociation constant of 5.9 x 10-8 M, whereas dsRBD1 binds RNA minimally. Binding analysis of 25-amino-acid peptides in TRBP and other related proteins showed that only one peptide in TRBP and one in Drosophila Staufen bind TAR and a GC-rich TAR-mimic RNA. Whereas a 25-mer peptide derived from dsRBD2 (TR5) bound TAR RNA, the equivalent peptide in dsRBD1 (TR6) did not. Molecular modelling indicates that this difference can mainly be ascribed to the replacement of Arg by His residues. Mutational analyses in homologous peptides also show the importance of residues K2 and L3. Analysis of 15-amino-acid peptides revealed that, in addition to TR13 (from TRBP dsRBD2), one peptide in S6 kinase has RNA-binding properties. On the basis of previous and the present results, we can define, in a broader context than that of TRBP, the main outlines of a modular KR-helix motif required for binding TAR. This structural motif exists independently from the dsRBD context and therefore has a modular function.

• 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
• Display abstract

• 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.

• Kim M, Hyun Park B, Lee Y
• Effects of terminal deletions in C5 protein on promoting RNase P catalysis.
• Biochem Biophys Res Commun. 2000; 268: 118-23
• Display abstract

• Deletion derivatives of C5 protein, the protein cofactor of Escherichia coli RNase P, were constructed as soluble MBP (maltose-binding protein) fusion proteins to assess the deletion effects on promoting RNase P catalysis and on binding to M1 RNA, the catalytic subunit of the enzyme. The C5 protein, with large terminal deletions, retained its promoting activity of RNase P catalysis under protein excess conditions in vitro. Some deletion derivatives complemented the temperature sensitive phenotype of E. coli A49 cells carrying the rnpA49 mutation. This ability also suggests that part of the C5 protein is enough to produce the catalytic activity of RNase P in vivo. Both the central conserved region, called the RNR motif, and the C-terminal region are essential for the binding of C5 protein to M1 RNA. Meanwhile, the N-terminal region contributes to promoting RNase P catalysis in ways other than binding to M1 RNA.

• Antson AA
• Single-stranded-RNA binding proteins.
• Curr Opin Struct Biol. 2000; 10: 87-94
• Display abstract

• 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.

• Raynal LC, Carpousis AJ
• Poly(A) polymerase I of Escherichia coli: characterization of the catalytic domain, an RNA binding site and regions for the interaction with proteins involved in mRNA degradation.
• Mol Microbiol. 1999; 32: 765-75
• Display abstract

• Poly(A) polymerase I (PAP I) of Escherichia coli is a member of the nucleotidyltransferase (Ntr) superfamily that includes the eukaryotic PAPs and all the known tRNA CCA-adding enzymes. Five highly conserved aspartic acids in the putative catalytic site of PAP I were changed to either alanine or proline, demonstrating their importance for polymerase activity. A glycine that is absolutely conserved in all Ntrs was also changed yielding a novel mutant protein in which ATP was wastefully hydrolysed in a primer-independent reaction. This is the first work to characterize the catalytic site of a eubacterial PAP and, despite the conservation of certain sequences, we predict that the overall architecture of the eukaryotic and eubacterial active sites is likely to be different. Binding sites for RNase E, a component of the RNA degradosome, and RNA were mapped by North-western and Far-western blotting using truncated forms of PAP I. Additional protein-protein interactions were detected between PAP I and CsdA, RhlE and SrmB, suggesting an unexpected connection between PAP I and these E. coli DEAD box RNA helicases. These results show that the functional organization of PAP I is similar to the eukaryotic PAPs with an N-terminal catalytic domain, a C-terminal RNA binding domain and sites for the interaction with other protein factors.

• 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.

• Krovat BC, Jantsch MF
• Comparative mutational analysis of the double-stranded RNA binding domains of Xenopus laevis RNA-binding protein A.
• J Biol Chem. 1996; 271: 28112-9
• Display abstract

• Xenopus laevis RNA-binding protein A is a ubiquitously expressed, double-stranded RNA-binding protein that is associated with the majority of cellular RNAs, ribosomal RNAs, and hnRNAs. X. laevis RNA-binding protein A contains three copies of the double-stranded RNA-binding domain (dsRBD) in tandem arrangement. Two of them, xl1 and xl2, belong to the type A group of dsRBDs that show strong homologies to the entire length of a defined consensus sequence. The xl3 domain, in contrast, is a type B dsRBD which only matches the basic C-terminal end of the dsRBD consensus sequence. Here we show that only xl2 but neither xl1 nor xl3 are able to bind double-stranded RNA substrates in vitro, suggesting that different dsRBD copies have varying RNA binding activities. By fine mapping mutagenesis of the isolated xl2 domain, we identified at least two central aromatic amino acids and a C-terminal alpha-helix that are indispensable for dsRNA binding. Furthermore, we show that different charge distributions within the C-terminal alpha-helices of xl1 and xl2 seem responsible for the different RNA binding behaviors of these two dsRBDs. Analyses of the RNA binding properties of constructs containing various combinations of different dsRBDs reveal that type A dsRBDs exhibit a cooperative binding effect, whereas type B dsRBDs show a rather low binding activity, thus contributing only to a minor extent to a stable RNA-protein interaction.

• Ribas JC, Fujimura T, Wickner RB
• A cryptic RNA-binding domain in the Pol region of the L-A double-stranded RNA virus Gag-Pol fusion protein.
• J Virol. 1994; 68: 6014-20
• Display abstract

• The Pol region of the Gag-Pol fusion protein of the L-A double-stranded (ds) RNA virus of Saccharomyces cerevisiae has (i) a domain essential for packaging viral positive strands, (ii) consensus amino acid sequence patterns typical of RNA-dependent RNA polymerases, and (iii) two single-stranded RNA binding domains. We describe here a third single-stranded RNA binding domain (Pol residues 374 to 432), which is unique in being cryptic. Its activity is revealed only after deletion of an inhibitory region C terminal to the binding domain itself. This cryptic RNA binding domain is necessary for propagation of M1 satellite dsRNA, but it is not necessary for viral particle assembly or for packaging of viral positive-strand single-stranded RNA. The cryptic RNA binding domain includes a sequence pattern common among positive-strand single-stranded RNA and dsRNA viral RNA-dependent RNA polymerases, suggesting that it has a role in RNA polymerase activity.

• Fukami-Kobayashi K, Go M
• [Molecular evolution of proteins with RNA-binding domains].
• Tanpakushitsu Kakusan Koso. 1994; 39: 2177-88

• Koonin EV, Deutscher MP
• RNase T shares conserved sequence motifs with DNA proofreading exonucleases.
• Nucleic Acids Res. 1993; 21: 2521-2

• Shirley BA, Stanssens P, Steyaert J, Pace CN
• Conformational stability and activity of ribonuclease T1 and mutants. Gln25----Lys, Glu58----Ala, and the double mutant.
• J Biol Chem. 1989; 264: 11621-5
• Display abstract

• Ribonuclease T1 (RNase T1) and mutants Gln25----Lys, Glu58----Ala, and the double mutant were prepared from a chemically synthesized gene, cloned and expressed in Escherichia coli. The wild-type RNase T1 prepared from the cloned gene was identical in every functional and physical property examined to RNase T1 prepared from Aspergillus oryzae. Urea and thermal unfolding experiments show that Gln25----Lys is 0.9 kcal/mol more stable and Glu58----Ala is 0.8 kcal/mol less stable than wild-type RNase T1. In the double mutant, these contributions cancel and the stability does not differ significantly from that of wild-type RNase T1. For the double mutant, the dependence of delta G on urea concentration is significantly greater than for wild-type RNase T1 or the single mutants. This suggests that the double mutant unfolds more completely in urea than the other proteins. The activity of Gln25----Lys is identical with that of wild-type RNase T1. The activities of Glu58----Ala and the double mutant are 7% of wild-type when GpC hydrolysis is measured (due to a 35-fold decrease in kcat), and 37% of wild-type when RNA hydrolysis is measured. Thus, Glu58 is important, but not essential to the activity of RNase T1.

• Apirion D, Watson N
• Unaltered stability of newly synthesized RNA in strains of Escherichia coli missing a ribonuclease specific for double-stranded RNA.
• Mol Gen Genet. 1975; 136: 317-26
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• Pairs of very closely related Escherichia coli strains were prepared, one having the wild-type allele for ribonuclease III, an enzyme which specifically degrades double-stranded RNA, and the other having a mutant RNase III allele. Growth and phage plating efficiency were compared in these strains. The RNase III+ strains grow better than the RNase III- strains and plate T7 and lambda phage better, but T4 plates with the same efficiency on both strains. On the other hand, the half lives of newly synthesized RNA as well as of functional beta-galactosidase mRNA are similar in both kind of strains. These two parameters, however, are significantly longer in both strains as compared to the original strain from which they were derived. Also, no difference in the differential induction of beta-galactosidase was observed between such strains. Thus, we have to conclude that either ribonuclease III does not play a significant role in the functioning and stability of newly synthesized mRNA, or that enough enzymatic activity was left, residual RNase III or some other enzyme to deal with double-stranded regions in the message.

• Sugita M, Fukuda N
• Functional analysis of chemical systems in vivo using a logical circuit equivalent. 3. Analysis using a digital circuit combined with an analogue computer.
• J Theor Biol. 1963; 5: 412-25

• GILBERT W
• Polypeptide synthesis in Escherichia coli. II. The polypeptide chain and S-RNA.
• J Mol Biol. 1963; 6: 389-403

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