Secondary literature sources for the ANTAR domain

For each of the hand selected primary papers associated with the ANTAR domain, 100 neighbouring papers are retrieved from PubMed. If one of these neighbouring papers is referenced by more than two primary papers, it is shown in the table below.

Action of an RNA site at a distance: role of the nut genetic signal in transcription antitermination by phage-lambda N gene product.

Whalen WA, Das A
New Biol. 1990; 2: 975-91

The N gene product of Escherichia coli phage lambda is a transcriptional activator that captures the host RNA polymerase and modifies it to a termination-resistant form, permitting gene expression in two large polycistronic operons of the phage genome. Antitermination in vitro requires at least one host factor called NusA, which directly binds the N protein as well as RNA polymerase, and also a transcribed cis-acting site known as nut, within which lies the hypothesized N-recognition signal, boxB. BoxB is an interrupted palindrome capable of forming a hairpin in the mRNA. Inhibition studies with complementary DNA oligonucleotides provide evidence for a direct role of the boxB hairpin in antitermination. Kinetic studies of transcript elongation reveal that the boxB hairpin does not induce an appreciable pause to hold polymerase captive for engagement by N and NusA. Moreover, the efficiency of antitermination remains virtually the same whether N and NusA are added early, prior to nut site transcription, or added later, after the polymerase has already transcribed past the nut site. After transcription of the nut site, RNA polymerase remains susceptible to modification by N and NusA for an appreciable amount of time and distance, and the nut site DNA becomes dispensable for this modification. These results lead to the hypothesis that the boxB RNA hairpin acts in a manner analogous to the DNA enhancers, binding N and mediating a productive polymerase-NusA-N interaction by mRNA looping.

NasT-mediated antitermination plays an essential role in the regulation of the assimilatory nitrate reductase operon in Azotobacter vinelandii.

Wang B, Pierson LS 3rd, Rensing C, Gunatilaka MK, Kennedy C
Appl Environ Microbiol. 2012; 78: 6558-67

Azotobacter vinelandii is a well-studied model system for nitrogen fixation in bacteria. Regulation of nitrogen fixation in A. vinelandii is independent of NtrB/NtrC, a conserved nitrogen regulatory system in proteobacteria. Previous work showed that an ntrC mutation in A. vinelandii resulted in a loss of induction of assimilatory nitrate and nitrite reductases encoded by the nasAB operon. In addition to NtrC, several other proteins, including NasT, a protein containing a potential RNA-binding domain ANTAR (AmiR and NasR transcription antitermination regulators), have been implicated in nasAB regulation. In this work, we characterize the sequence upstream of nasA and identify several DNA sequence elements, including two potential NtrC binding sites and a putative intrinsic transcriptional terminator upstream of nasA that are potentially involved in nasAB regulation. Our analyses confirm that the nasAB promoter, P(nasA), is under NtrC control. However, unlike NtrC-regulated promoters in enteric bacteria, P(nasA) shows high activity in the presence of ammonium; in addition, the P(nasA) activity is altered in the nifA gene mutation background. We discuss the implication of these results on NtrC-mediated regulation in A. vinelandii. Our study provides direct evidence that induction of nasAB is regulated by NasT-mediated antitermination, which occurs within the leader region of the operon. The results also support the hypothesis that NasT binds the promoter proximal hairpin of nasAB for its regulatory function, which contributes to the understanding of the regulatory mechanism of ANTAR-containing antiterminators.

Assembly of the N-dependent antitermination complex of phage lambda: NusA and RNA bind independently to different unfolded domains of the N protein.

Van Gilst MR, von Hippel PH
J Mol Biol. 1997; 274: 160-73

The N protein of bacteriophage lambda activates expression of the delayed early genes of this phage by modifying RNA polymerase (RNAP) into a form that is resistant to termination signals. N binds to the boxB hairpin that forms in the nascent RNA transcript upon transcription of the nut regulatory element, and then interacts with RNAP by RNA looping. The binding of the N-boxB subassembly to the transcription complex is further stabilized by interaction with the Escherichia coli NusA protein. N, free in solution, exists as an unfolded protein that becomes partially structured upon binding specifically to boxB RNA. Because NusA does not assist in antitermination unless N is specifically bound to boxB, we have asked whether the structural change induced by binding to boxB affects the interaction of N with NusA. Using fluorescence spectroscopy, we have measured the affinity of N for NusA in the presence and absence of boxB RNA. We find that NusA binds to the unfolded N protein with a dissociation constant (Kd) of approximately 70 nM, and although N undergoes a significant structural change upon binding to boxB, the binding affinity of NusA for a N protein complexed with boxB is not altered. We have also shown that the boxA element of nut does not affect NusA binding to N-boxB. These results demonstrate that the interaction of N with NusA is independent of RNA binding, arguing that NusA must interact with an unfolded region of the polypeptide that remains unstructured even when N binds to boxB RNA. To further establish this point we isolated a truncated peptide containing the amino-terminal 36 residues of the N protein. Binding of boxB RNA to this peptide showed that all of the structural change in N that occurs upon binding to boxB RNA is localized within the amino-terminal 36 residues of N, therefore the C terminus of N, including the regions necessary for NusA binding and RNAP activation, remains unfolded when the full length N binds to boxB RNA. Thus it appears that N can be described as an unfolded multi-domain protein that becomes ordered in a modular fashion as it encounters its various binding partners within the N-dependent antitermination complex.

Inhibition of the B. subtilis regulatory protein TRAP by the TRAP-inhibitory protein, AT.

Valbuzzi A, Yanofsky C
Science. 2001; 293: 2057-9

An anti-TRAP (AT) protein, a factor of previously unknown function, conveys the metabolic signal that the cellular transfer RNA for tryptophan (tRNATrp) is predominantly uncharged. Expression of the operon encoding AT is induced by uncharged tRNATrp. AT associates with TRAP, the trp operon attenuation protein, and inhibits its binding to its target RNA sequences. This relieves TRAP-mediated transcription termination and translation inhibition, increasing the rate of tryptophan biosynthesis. AT binds to TRAP primarily when it is in the tryptophan-activated state. The 53-residue AT polypeptide is homologous to the zinc-binding domain of DnaJ. The mechanisms regulating tryptophan biosynthesis in Bacillus subtilis differ from those used by Escherichia coli.

The anti-trp RNA-binding attenuation protein (Anti-TRAP), AT, recognizes the tryptophan-activated RNA binding domain of the TRAP regulatory protein.

Valbuzzi A, Gollnick P, Babitzke P, Yanofsky C
J Biol Chem. 2002; 277: 10608-13

In Bacillus subtilis, the trp RNA-binding attenuation protein (TRAP) regulates expression of genes involved in tryptophan metabolism in response to the accumulation of l-tryptophan. Tryptophan-activated TRAP negatively regulates expression by binding to specific mRNA sequences and either promoting transcription termination or blocking translation initiation. Conversely, the accumulation of uncharged tRNA(Trp) induces synthesis of an anti-TRAP protein (AT), which forms a complex with TRAP and inhibits its activity. In this report, we investigate the structural features of TRAP required for AT recognition. A collection of TRAP mutant proteins was examined that were known to be partially or completely defective in tryptophan binding and/or RNA binding. Analyses of AT interactions with these proteins were performed using in vitro transcription termination assays and cross-linking experiments. We observed that TRAP mutant proteins that had lost the ability to bind RNA were no longer recognized by AT. Our findings suggest that AT acts by competing with messenger RNA for the RNA binding domain of TRAP. B. subtilis AT was also shown to interact with TRAP proteins from Bacillus halodurans and Bacillus stearothermophilus, implying that the structural elements required for AT recognition are conserved in the TRAP proteins of these species. Analyses of AT interaction with B. stearothermophilus TRAP at 60 degrees C demonstrated that AT is active at this elevated temperature.

Novel regulatory cascades controlling expression of nitrogen-fixation genes in Geobacter sulfurreducens.

Ueki T, Lovley DR
Nucleic Acids Res. 2010; 38: 7485-99

Geobacter species often play an important role in bioremediation of environments contaminated with metals or organics and show promise for harvesting electricity from waste organic matter in microbial fuel cells. The ability of Geobacter species to fix atmospheric nitrogen is an important metabolic feature for these applications. We identified novel regulatory cascades controlling nitrogen-fixation gene expression in Geobacter sulfurreducens. Unlike the regulatory mechanisms known in other nitrogen-fixing microorganisms, nitrogen-fixation gene regulation in G. sulfurreducens is controlled by two two-component His-Asp phosphorelay systems. One of these systems appears to be the master regulatory system that activates transcription of the majority of nitrogen-fixation genes and represses a gene encoding glutamate dehydrogenase during nitrogen fixation. The other system whose expression is directly activated by the master regulatory system appears to control by antitermination the expression of a subset of the nitrogen-fixation genes whose transcription is activated by the master regulatory system and whose promoter contains transcription termination signals. This study provides a new paradigm for nitrogen-fixation gene regulation.

Genetic analysis of bacteriophage lambdaN-dependent antitermination suggests a possible role for the RNA polymerase alpha subunit in facilitating specific functions of NusA and NusE.

Szalewska-Palasz A, Strzelczyk B, Herman-Antosiewicz A, Wegrzyn G, Thomas MS
Arch Microbiol. 2003; 180: 161-8

A role for the Escherichia coli RNA polymerase alpha subunit in transcription antitermination dependent on bacteriophage lambda N protein has been previously inferred from the isolation of rpoA mutants that alter the efficiency of this process. This report describes studies on the efficiency of N-dependent transcription antitermination in a strain containing the rpoA341 mutation, which interferes with this process. The effect of mutations in genes coding for different Nus factors and/or plasmids overexpressing nus genes on bacteriophage lambda development in an E. coli rpoA341 host was examined. In addition, the effect of overproduction of the N protein in these genetic backgrounds was assessed. Analogous bacterial strains were employed to measure the efficiency of the antitermination process using the lacZ reporter gene under control of the lambda p(R) promoter, and containing the phage nutR region and the t( R1) terminator between the promoter and lacZ. The experimental results suggest interactions between components of the N-antitermination complex, which have been established biochemically, as well as additional functional relationships within the complex. Furthermore, the results indicate that amino acid substitution in the alpha subunit C-terminal domain encoded by the rpoA341 mutation may specifically disrupt the function of the NusA and NusE proteins. During this analysis, it was also found that the E. coli nusA1 mutant exhibits a conditional lethal phenotype.

Rrp1, a cyclic-di-GMP-producing response regulator, is an important regulator of Borrelia burgdorferi core cellular functions.

Rogers EA, Terekhova D, Zhang HM, Hovis KM, Schwartz I, Marconi RT
Mol Microbiol. 2009; 71: 1551-73

Two-component systems (TCS) are universal among bacteria and play critical roles in gene regulation. Our understanding of the contributions of TCS in the biology of the Borrelia is just now beginning to develop. Borrelia burgdorferi, a causative agent of Lyme disease, harbours a TCS comprised of open reading frames (ORFs) BB0419 and BB0420. BB0419 encodes a response regulator designated Rrp1, and BB0420 encodes a hybrid histidine kinase-response regulator designated Hpk1. Rrp1, which contains a conserved GGDEF domain, undergoes phosphorylation and produces the secondary messenger, cyclic diguanylate (c-di-GMP), a critical signaling molecule in numerous organisms. However, the regulatory role of the Rrp1-Hpk1 TCS and c-di-GMP signaling in Borrelia biology are unexplored. In this study, the distribution, conservation, expression and potential global regulatory capability of Rrp1 were assessed. rrp1 was found to be universal and highly conserved among isolates, co-transcribed with hpk1, constitutively expressed during in vitro cultivation, and significantly upregulated upon tick feeding. Allelic exchange replacement and microarray analyses revealed that the Rrp1 regulon consists of a large number of genes encoded by the core Borrelia genome (linear chromosome, linear plasmid 54 and circular plasmid 26) that encode for proteins involved in central metabolic processes and virulence mechanisms including immune evasion.

Bacteriophage lambda N-dependent transcription antitermination. Competition for an RNA site may regulate antitermination.

Patterson TA, Zhang Z, Baker T, Johnson LL, Friedman DI, Court DL
J Mol Biol. 1994; 236: 217-28

Bacteriophage lambda controls the expression of its early genes in a temporal manner by a series of transcription termination and antitermination events. This antitermination requires the lambda N protein as well as host proteins called Nus, and cis-acting sites called nut. Following transcription of the nut site, N and Nus proteins bind to the nut RNA and modify the transcription complex to a termination-resistant form. The nut site is a composite of at least two components; one is the boxB hairpin structure which interacts with N. The other is boxA, a nine-nucleotide sequence upstream of boxB. To understand more about the formation of the antitermination complex, we have characterized the effect of point mutations in and deletions of boxA on antitermination. Point mutations in boxA were found to either enhance or reduce N-mediated antitermination. Several boxA deletions, on the other hand, had little effect on antitermination other than to eliminate the requirement for the NusB host protein. To explain these observations, we propose that at least two factors compete to interact with boxA, NusB and an inhibitor of the antitermination reaction. In addition, we propose that NusB is required to prevent the inhibitor from binding at boxA. The results with various nusB and boxA mutations can be explained by this model of competition between NusB and an inhibitor for boxA RNA.

Recognition of boxA antiterminator RNA by the E. coli antitermination factors NusB and ribosomal protein S10.

Nodwell JR, Greenblatt J
Cell. 1993; 72: 261-8

The boxA sequences of the E. coli ribosomal RNA (rrn) operons are sufficient to cause RNA polymerase to read through Rho-dependent transcriptional terminators. We show that a complex of the transcription antitermination factors NusB and ribosomal protein S10 interacts specifically with boxA RNA. Neither NusB nor S10 binds boxA RNA on its own, and neither NusA nor NusG affects the interaction of the NusB-S10 complex with boxA RNA. Mutations in boxA that impair its antitermination activity compromise its interaction with NusB and S10, suggesting that ribosomal protein S10 regulates the synthesis of ribosomal RNA in bacteria. RNA containing the closely related boxA sequence from the bacteriophage lambda nutR site is not stably bound by NusB and S10. This probably explains why antitermination in phage lambda depends on the phage lambda N protein and the boxB component of the nut site, in addition to boxA.

PIRSF family classification system for protein functional and evolutionary analysis.

Nikolskaya AN, Arighi CN, Huang H, Barker WC, Wu CH
Evol Bioinform Online. 2006; 2: 197-209

The PIRSF protein classification system (http://pir.georgetown.edu/pirsf/) reflects evolutionary relationships of full-length proteins and domains. The primary PIRSF classification unit is the homeomorphic family, whose members are both homologous (evolved from a common ancestor) and homeomorphic (sharing full-length sequence similarity and a common domain architecture). PIRSF families are curated systematically based on literature review and integrative sequence and functional analysis, including sequence and structure similarity, domain architecture, functional association, genome context, and phyletic pattern. The results of classification and expert annotation are summarized in PIRSF family reports with graphical viewers for taxonomic distribution, domain architecture, family hierarchy, and multiple alignment and phylogenetic tree. The PIRSF system provides a comprehensive resource for bioinformatics analysis and comparative studies of protein function and evolution. Domain or fold-based searches allow identification of evolutionarily related protein families sharing domains or structural folds. Functional convergence and functional divergence are revealed by the relationships between protein classification and curated family functions. The taxonomic distribution allows the identification of lineage-specific or broadly conserved protein families and can reveal horizontal gene transfer. Here we demonstrate, with illustrative examples, how to use the web-based PIRSF system as a tool for functional and evolutionary studies of protein families.

Members of the IclR family of bacterial transcriptional regulators function as activators and/or repressors.

Molina-Henares AJ, Krell T, Eugenia Guazzaroni M, Segura A, Ramos JL
FEMS Microbiol Rev. 2006; 30: 157-86

Members of the IclR family of regulators are proteins with around 250 residues. The IclR family is best defined by a profile covering the effector binding domain. This is supported by structural data and by a number of mutants showing that effector specificity lies within a pocket in the C-terminal domain. These regulators have a helix-turn-helix DNA binding motif in the N-terminal domain and bind target promoters as dimers or as a dimer of dimers. This family comprises regulators acting as repressors, activators and proteins with a dual role. Members of the IclR family control genes whose products are involved in the glyoxylate shunt in Enterobacteriaceae, multidrug resistance, degradation of aromatics, inactivation of quorum-sensing signals, determinants of plant pathogenicity and sporulation. No clear consensus exists on the architecture of DNA binding sites for IclR activators: the MhpR binding site is formed by a 15-bp palindrome, but the binding sites of PcaU and PobR are three perfect 10-bp sequence repetitions forming an inverted and a direct repeat. IclR-type positive regulators bind their promoter DNA in the absence of effector. The mechanism of repression differs among IclR-type regulators. In most of them the binding sites of RNA polymerase and the repressor overlap, so that the repressor occludes RNA polymerase binding. In other cases the repressor binding site is distal to the RNA polymerase, so that the repressor destabilizes the open complex.

Function of transcription cleavage factors GreA and GreB at a regulatory pause site.

Marr MT, Roberts JW
Mol Cell. 2000; 6: 1275-85

Gre proteins of prokaryotes, and SII proteins of eukaryotes and archaea, are transcription elongation factors that promote an endogenous transcript cleavage activity of RNA polymerases; this process promotes elongation through obstructive regions of DNA, including transcription pauses that act as sites of genetic regulation. We show that a regulatory pause in the early part of the late gene operon of bacteriophage lambda is subject to such cleavage and resynthesis. In cells lacking the cleavage factors GreA and GreB, the pause is prolonged, and RNA polymerase occupies a variant position at the pause site. Furthermore, GreA and GreB are required to mediate efficient function of the lambda gene Q antiterminator at this site. Thus, cleavage factors are necessary for the natural progression of RNA polymerase in vivo.

Specific interaction of the RNA-binding domain of the bacillus subtilis transcriptional antiterminator GlcT with its RNA target, RAT.

Langbein I, Bachem S, Stulke J
J Mol Biol. 1999; 293: 795-805

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.

A bacterial transcription terminator with inefficient molecular motor action but with a robust transcription termination function.

Kalarickal NC, Ranjan A, Kalyani BS, Wal M, Sen R
J Mol Biol. 2010; 395: 966-82

Molecular motors such as helicases/translocases are capable of translocating along the single-stranded nucleic acids and unwinding DNA or RNA duplex substrates using the energy derived from their ATPase activity. The bacterial transcription terminator, Rho, is a hexameric helicase and releases RNA from the transcription elongation complexes by an unknown mechanism. It has been proposed, but not directly demonstrated, that kinetic energy obtained from its molecular motor action (helicase/translocase activities) is instrumental in dissociating the transcription elongation complex. Here we report a hexameric Rho analogue (Rv1297, M. tb. Rho) from Mycobacterium tuberculosis having poor RNA-dependent ATP hydrolysis and inefficient DNA-RNA unwinding activities. However, compared to Escherichia coli Rho, it exhibited very robust and earlier transcription termination from the elongation complexes of E. coli RNA polymerase. Bicyclomycin, an inhibitor of ATPase as well as RNA release activities of E. coli Rho, inhibited the ATPase activity of M. tb. Rho with comparable efficiency but was not efficient in inhibiting its transcription termination function. Unlike E. coli Rho, M. tb. Rho was capable of releasing RNA in the presence of nonhydrolyzable analogues of ATP quite efficiently. Also, this termination function most likely does not require NusG, an RNA-release facilitator, as this Rho was incapable of binding to NusG either of M. tb. (Rv0639) or E. coli. These results strongly suggest that the ATPase activity of M. tb. Rho is uncoupled from its transcription termination function and this function may not be dependent on its helicase/translocase activity.

The crystal structure of NusB from Mycobacterium tuberculosis.

Gopal B, Haire LF, Cox RA, Jo Colston M, Major S, Brannigan JA, Smerdon SJ, Dodson G
Nat Struct Biol. 2000; 7: 475-8

Both prokaryotes and eukaryotes regulate transcription through mechanisms that suppress termination signals. An antitermination mechanism was first characterized in bacteriophage lambda. Bacteria have analogous machinery that regulates ribosomal RNA transcription and employs host factors, called the N-utilizing (where N stands for the phage lambda N protein) substances (Nus), NusA, NusB, NusE and NusG. Here we report the crystal structure of NusB from Mycobacterium tuberculosis, the bacterium that causes tuberculosis in humans. This molecule shares a similar tertiary structure with the related Escherichia coli protein but adopts a different quaternary organization. We show that, unlike the E. coli homolog, M. tuberculosis NusB is dimeric both in solution and in the crystal. These data help provide a framework for understanding the structural and biological function of NusB in the prokaryotic transcriptional antitermination complex.

Regulation of the Bacillus subtilis trp operon by an RNA-binding protein.

Gollnick P
Mol Microbiol. 1994; 11: 991-7

The Bacillus subtilis tryptophan (trpEDCFBA) operon is regulated by transcription attenuation. Transcription is controlled by two alternative RNA secondary structures, which form in the leader transcript. In the presence of L-tryptophan, a transcription terminator forms and the operon is not expressed, whereas in the absence of tryptophan, an antiterminator structure forms allowing transcription of the operon. The mechanism of selection between these alternative structures involves a trans-acting RNA-binding regulatory protein. This protein is the product of the mtrB gene and is called TRAP for trp attenuation protein. TRAP has been shown to bind specifically to trp leader RNA, and to cause transcription of the trp operon to terminate in the leader region. The model for regulation suggests that in the presence of tryptophan, TRAP binds to the leader RNA and induces formation of the transcription terminator structure, whereas in the absence of tryptophan, the protein does not bind and the antiterminator is formed.

Ethanolamine activates a sensor histidine kinase regulating its utilization in Enterococcus faecalis.

Del Papa MF, Perego M
J Bacteriol. 2008; 190: 7147-56

Enterococcus faecalis is a gram-positive commensal bacterium of the human intestinal tract. Its opportunistic pathogenicity has been enhanced by the acquisition of multiple antibiotic resistances, making the treatment of enterococcal infections an increasingly difficult problem. The extraordinary capacity of this organism to colonize and survive in a wide variety of ecological niches is attributable, at least in part, to signal transduction pathways mediated by two-component systems (TCS). Here, the ability of E. faecalis to utilize ethanolamine as the sole carbon source is shown to be dependent upon the RR-HK17 (EF1633-EF1632) TCS. Ethanolamine is an abundant compound in the human intestine, and thus, the ability of bacteria to utilize it as a source of carbon and nitrogen may provide an advantage for survival and colonization. Growth of E. faecalis in a synthetic medium with ethanolamine was abolished in the response regulator RR17 mutant strain. Transcription of the response regulator gene was induced by the presence of ethanolamine. Ethanolamine induced a 15-fold increase in the rate of autophosphorylation in vitro of the HK17 sensor histidine kinase, indicating that this is the ligand recognized by the sensor domain of the kinase. These results assign a role to the RR-HK17 TCS as coordinator of the enterococcal response to specific nutritional conditions existing at the site of bacterial invasion, the intestinal tract of an animal host.

RNA recognition by transcriptional antiterminators of the BglG/SacY family: functional and structural comparison of the CAT domain from SacY and LicT.

Declerck N, Vincent F, Hoh F, Aymerich S, van Tilbeurgh H
J Mol Biol. 1999; 294: 389-402

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.

RNA recognition by transcriptional antiterminators of the BglG/SacY family: mapping of SacY RNA binding site.

Declerck N, Minh NL, Yang Y, Bloch V, Kochoyan M, Aymerich S
J Mol Biol. 2002; 319: 1035-48

Transcriptional antiterminators of the BglG/SacY family are bacterial regulatory proteins able to prevent the premature arrest of transcription through specific binding to a ribonucleic antiterminator (RAT) sequence. The RNA recognition module of these regulators is made of the 55-amino acid long N-terminal domain which can by itself promote efficient antitermination activity in vivo and RNA binding in vitro. The structure of this domain, which was called CAT for co-antiterminator, has first been determined for SacY from Bacillus subtilis and the putative surface contacting RNA has been defined by NMR footprinting. Here we have performed a genetic mapping of the SacY-CAT RNA binding site by substituting 24 amino acid residues including those previously identified by NMR, the highly conserved residues in the 55 homologous antiterminators recognised in the databases and all the positively charged residues. A total of 57 SacY-CAT variants have been constructed and tested in vivo for their antitermination efficiency. A few of these variants were then purified in order to analyse their RNA binding properties by surface plasmon resonance and to check their structural integrity by NMR. The present study validates and clarifies the RNA interacting surface previously mapped by NMR. The residues that are the most intolerant to substitutions, Asn8, His9, Asn10, Gly25, Gly27, and Phe30, are aligned across the CAT dimer interface and form the core of the RNA binding site. Three highly conserved residues stand outside the interaction surface but are essential for maintaining the CAT dimeric structure (Phe47) or may play an important functional role in the full length protein (Glu20 and Lys32). Interestingly, none of the twelve positively charged residues of SacY-CAT are crucial for the antitermination activity. By replacing three Lys residues and combining the Ala26-->Arg mutation that significantly enhanced the affinity for RNA, we engineered a SacY-CAT variant that should be suitable for NMR study of the complex.