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Tina M Henkin - One of the best experts on this subject based on the ideXlab platform.
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non conserved residues in clostridium acetobutylicum trnaala contribute to trna tuning for efficient Antitermination of the alas t box riboswitch
Life, 2015Co-Authors: Frank J Grundy, Tina M HenkinAbstract:The T box riboswitch regulates expression of amino acid-related genes in Gram-positive bacteria by monitoring the aminoacylation status of a specific tRNA, the binding of which affects the folding of the riboswitch into mutually exclusive terminator or antiterminator structures. Two main pairing interactions between the tRNA and the leader RNA have been demonstrated to be necessary, but not sufficient, for efficient Antitermination. In this study, we used the Clostridium acetobutylicum alaS gene, which encodes alanyl-tRNA synthetase, to investigate the specificity of the tRNA response. We show that the homologous C. acetobutylicum tRNAAla directs Antitermination of the C. acetobutylicum alaS gene in vitro, but the heterologous Bacillus subtilis tRNAAla (with the same anticodon and acceptor end) does not. Base substitutions at positions that vary between these two tRNAs revealed synergistic and antagonistic effects. Variation occurs primarily at positions that are not conserved in tRNAAla species, which indicates that these non-conserved residues contribute to optimal Antitermination of the homologous alaS gene. This study suggests that elements in tRNAAla may have coevolved with the homologous alaS T box leader RNA for efficient Antitermination.
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kinetic analysis of trna directed transcription Antitermination of the bacillus subtilis glyqs gene in vitro
Journal of Bacteriology, 2004Co-Authors: Frank J Grundy, Tina M HenkinAbstract:Binding of uncharged tRNA to the nascent transcript promotes readthrough of a leader region transcription termination signal in genes regulated by the T box transcription Antitermination mechanism. Each gene in the T box family responds independently to its cognate tRNA, with specificity determined by base pairing of the tRNA to the leader at the anticodon and acceptor ends of the tRNA. tRNA binding stabilizes an antiterminator element in the transcript that sequesters sequences that participate in formation of the terminator helix. tRNAGly-dependent Antitermination of the Bacillus subtilis glyQS leader was previously demonstrated in a purified in vitro assay system. This assay system was used to investigate the kinetics of transcription through the glyQS leader and the effect of tRNA and transcription elongation factors NusA and NusG on transcriptional pausing and Antitermination. Several pause sites, including a major site in the loop of stem III of the leader, were identified, and the effect of modulation of pausing on Antitermination efficiency was analyzed. We found that addition of tRNAGly can promote Antitermination as long as the tRNA is added before the majority of the transcription complexes reach the termination site, and variations in pausing affect the requirements for timing of tRNA addition.
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trna requirements for glyqs Antitermination a new twist on trna
RNA, 2003Co-Authors: Mary R Yousef, Frank J Grundy, Tina M HenkinAbstract:Transcription Antitermination of the Bacillus subtilis glyQS gene, a member of the T box gene regulation family, can be induced during in vitro transcription in a minimal system using purified B. subtilis RNA polymerase by the addition of unmodified T7 RNA polymerase-transcribed tRNAGly. Antitermination was previously shown to depend on base-pairing between the glyQS leader and the tRNA at the anticodon and acceptor ends. In this study, variants of tRNAGly were generated to identify additional tRNA elements required for Antitermination activity, and to determine the effect of structural changes in the tRNA. We find that additions to the 3′ end of the tRNA blocked Antitermination, in agreement with the prediction that uncharged tRNA is the effector in vivo, whereas insertion of 1 nucleotide between the acceptor stem and the 3′ UCCA residues had no effect. Disruption of the D-loop/T-loop tertiary interaction inhibited Antitermination function, as was previously demonstrated for tRNATyr-directed Antitermination of the B. subtilis tyrS gene in vivo. Insertion of a single base pair in the anticodon stem was tolerated, whereas further insertions abolished Antitermination. However, we find that major alterations in the length of the acceptor stem are tolerated, and the insertions exhibited a pattern of periodicity suggesting that there is face-of-the-helix dependence in the positioning of the unpaired UCCA residues at the 3′ end of the tRNA for interaction with the antiterminator bulge and Antitermination.
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trna mediated transcription Antitermination in vitro codon anticodon pairing independent of the ribosome
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Frank J Grundy, Wade C Winkler, Tina M HenkinAbstract:Uncharged tRNA acts as the effector for transcription Antitermination of genes in the T box family in Bacillus subtilis and other Gram-positive bacteria. Genetic studies suggested that expression of these genes is induced by stabilization of an antiterminator element in the leader RNA of each target gene by the cognate uncharged tRNA. The specificity of the tRNA response is dependent on a single codon in the leader, which was postulated to pair with the anticodon of the corresponding tRNA. It was not known whether the leader RNA–tRNA interaction requires additional factors. We show here that tRNA-dependent Antitermination occurs in vitro in a purified transcription system, in the absence of ribosomes or accessory factors, demonstrating that the RNA–RNA interaction is sufficient to control gene expression by Antitermination. The tRNA response exhibits similar specificity in vivo and in vitro, and the Antitermination reaction in vitro is independent of NusA and functions with either B. subtilis or Escherichia coli RNA polymerase.
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analysis of cis acting sequence and structural elements required for Antitermination of the bacillus subtilis tyrs gene
Molecular Microbiology, 1997Co-Authors: Sean M Rollins, Frank J Grundy, Tina M HenkinAbstract:Summary The Bacillus subtilis tyrS gene belongs to the T box family of aminoacyl-tRNA synthetase and amino acid biosynthesis genes, which are regulated by a common mechanism of transcriptional Antitermination. Each gene is induced by specific amino acid limitation; the uncharged cognate tRNA is the effector inducing transcription of the full-length message. The leader regions of the genes in this family share a number of conserved primary sequence and secondary structural elements, the functions of which are unknown. In this study, we examine these regions and report the effects of mutations in several of these elements. In addition, two alternative basepairings in the F box region were found to be necessary for tyrS Antitermination.
David I Friedman - One of the best experts on this subject based on the ideXlab platform.
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evidence that the promoter can influence assembly of Antitermination complexes at downstream rna sites
Journal of Bacteriology, 2006Co-Authors: Ying Zhou, Eric R. Olson, Mark A Mozola, Karla S Henthorn, Susan Brown, Gary N Gussin, David I FriedmanAbstract:The N protein of phage λ acts with Escherichia coli Nus proteins at RNA sites, NUT, to modify RNA polymerase (RNAP) to a form that overrides transcription terminators. These interactions have been thought to be the primary determinants of the effectiveness of N-mediated Antitermination. We present evidence that the associated promoter, in this case the λ early PR promoter, can influence N-mediated modification of RNAP even though modification occurs at a site (NUTR) located downstream of the intervening cro gene. As predicted by genetic analysis and confirmed by in vivo transcription studies, a combination of two mutations in PR, at positions −14 and −45 (yielding PR-GA), reduces effectiveness of N modification, while an additional mutation at position −30 (yielding PR-GCA) suppresses this effect. In vivo, the level of PR-GA-directed transcription was twice as great as the wild-type level, while transcription directed by PR-GCA was the same as that directed by the wild-type promoter. However, the rate of open complex formation at PR-GA in vitro was roughly one-third the rate for wild-type PR. We ascribe this apparent discrepancy to an effect of the mutations in PR-GCA on promoter clearance. Based on the in vivo experiments, one plausible explanation for our results is that increased transcription can lead to a failure to form active Antitermination complexes with NUT RNA, which, in turn, causes failure to read through downstream termination sites. By blocking Antitermination and thus expression of late functions, the effect of increased transcription through nut sites could be physiologically important in maintaining proper regulation of gene expression early in phage development.
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analyzing transcription Antitermination in lambdoid phages encoding toxin genes
Methods in Enzymology, 2003Co-Authors: Melody N Neely, David I FriedmanAbstract:Publisher Summary This chapter analyzes the transcription of Antitermination in lambdoid phages encoding toxin genes. Lambdoid phages carry the genes encoding Shiga toxin (Stx A and B): most commonly, the genes encoding the two subunits of either the stx1 or stx2 genes. The two best-studied stx-carrying phages are Stx1 phage H-19B and Stx2 phage 933W. Four methods of analysis were used to determine whether H-19B has an Antitermination system similar to that of λ and assess its specific mechanism of action: DNA sequence determination of regions of the H-19B genome that may play a role in Antitermination based on studies of λ N-mediated Antitermination; growth of H-19B in E. coli mutants defective for λ Antitermination; construction and analysis of H-19B phage mutants; and reporter constructs designed to study Antitermination mediated by the H-19B N protein. Analysis of H-19B Q-mediated Antitermination involved similar methods as those employed in analysis of N-mediated Antitermination: sequence determination of the late regulatory region of H-19B, which includes the Q gene, the Prʹ promoter with its associated qut site, and the t R ʹ terminator, as well as stx and lysis genes; phage mutants; and reporter–terminator constructs to measure factors required for Antitermination at downstream terminators.
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requirement for nusg for transcription Antitermination in vivo by the λ n protein
Journal of Bacteriology, 2002Co-Authors: Ying Zhou, Donald L Court, Joshua J Filter, Max E Gottesman, David I FriedmanAbstract:Transcription Antitermination by the bacteriophage λ N protein is stimulated in vitro by the Escherichia coli NusG protein. Earlier work suggested that NusG was not required for N activity in vivo. Here we present evidence that NusG also stimulates N-mediated transcription Antitermination in intact cells.
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interactions of an arg rich region of transcription elongation protein nusa with nut rna implications for the order of assembly of the lambda n Antitermination complex in vivo
Journal of Molecular Biology, 2001Co-Authors: Ying Zhou, Eric R. Olson, Jack Greenblatt, Jeremy Mogridge, Yuentsu N Yu, David I FriedmanAbstract:Abstract The E. coli NusA transcription elongation protein (NusA Ec ), identified because of its requirement for transcription Antitermination by the N protein, has an Arg-rich S1 RNA-binding domain. A complex of N and NusA with other host factors binding at NUT sites in the RNA renders RNA polymerase termination-resistant. An E. coli haploid for nusA944 , having nine different codons replacing four normally found in the Arg-rich region, is defective in support of N action. Another variant, haploid for the nusAR199A allele, with a change in a highly conserved Arg codon in the S1 domain, effectively supports N-mediated Antitermination. However, nusAR199A is recessive to nusA944 , while nusA Ec is dominant to nusA944 for support of N-mediated Antitermination, suggesting a competition between NusA944 and NusAR199A during complex formation. Complex formation with the variant NusA proteins was assessed by mobility gel shifts. NusAR199A, unlike NusA Ec and NusA944, fails to form a complex with N and NUT RNA. However, while NusAR199A, like wild-type NusA, forms an enlarged complex with NUT RNA, N, RNA polymerase, and other host proteins required for efficient N-mediated Antitermination, NusA944 does not form this enlarged complex. Consistent with the in vivo results, NusA944 prevents NusAR199A but not NusA Ec from forming the enlarged complex. The simplest conclusion from these dominance studies is that in the formation of the complete active Antitermination complex in vivo , NusA and N binding to the newly synthesized NUT RNA precedes addition of the other factors. Alternative less effective routes to the active complex that allows bypass of this preferred pathway may also exist.
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The alpha subunit of RNA polymerase and transcription Antitermination
Molecular Microbiology, 1996Co-Authors: A. T. Schauer, Donald L Court, Sheau Wei C. Cheng, Chuanhai Zheng, Linda St. Pierre, Diane M. Alessi, Debra L. Hidayetoglu, Nina Costantino, David I FriedmanAbstract:Summary The N gene product of coliphage , with a number of host proteins (Nus factors), regulates phage gene expression by modifying RNA polymerase to a form that overrides transcription-termination signals. Mutations in host nus genes diminish this N-mediated Antitermination. Here, we report the isolation and characterization of the rpoAD305E mutation, a single amino acid change in the carboxy terminal domain (CTD) of the subunit of RNA polymerase, that enhances N-mediated Antitermination. A deletion of the 3 terminus of rpoA, resulting in the expression of an subunit missing the CTD, also enhances Nmediated Antitermination and, similar to rpoAD305E, suppresses the effect of nus mutations. Thus, the N‐ Nus complex may be affected through contacts with the CTD of the subunit of RNA polymerase, as is a group of regulatory proteins that influences initiation of transcription. What distinguishes our findings on the N‐Nus complex from those of previous studies with transcription proteins is that all of the regulators characterized in those studies bind DNA and influence transcription initiation; whereas the N‐Nus complex binds RNA and affects transcription elongation. A screen of some previously identified rpoA mutations that influence transcription activators revealed only one other amino acid change, L290H, in the CTD of the subunit, that influences Antitermination. Although our results provide evidence that interactions of the subunit of RNA polymerase must be considered in forming models of transcription Antitermination, they do not provide information as to whether the interactions of that ultimately influence Antitermination occur during initiation or during elongation of transcription.
Donald L Court - One of the best experts on this subject based on the ideXlab platform.
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structural basis for rna recognition by nusb and nuse in the initiation of transcription Antitermination
Nucleic Acids Research, 2011Co-Authors: Jason Stagno, Donald L Court, Amanda S Altieri, Mikhail Bubunenko, Sergey G Tarasov, Jess Li, Andrew R Byrd, Xinhua JiAbstract:Processive transcription Antitermination requires the assembly of the complete Antitermination complex, which is initiated by the formation of the ternary NusB–NusE–BoxA RNA complex. We have elucidated the crystal structure of this complex, demonstrating that the BoxA RNA is composed of 8 nt that are recognized by the NusB–NusE heterodimer. Functional biologic and biophysical data support the structural observations and establish the relative significance of key protein–protein and protein–RNA interactions. Further crystallographic investigation of a NusB–NusE–dsRNA complex reveals a heretofore unobserved dsRNA binding site contiguous with the BoxA binding site. We propose that the observed dsRNA represents BoxB RNA, as both single-stranded BoxA and double-stranded BoxB components are present in the classical lambda Antitermination site. Combining these data with known interactions amongst Antitermination factors suggests a specific model for the assembly of the complete Antitermination complex.
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structural and functional analysis of the e coli nusb s10 transcription Antitermination complex
Molecular Cell, 2009Co-Authors: Hehsuan Hsiao, Donald L Court, Mikhail Bubunenko, Max E Gottesman, G Weber, Henning Urlaub, Markus C WahlAbstract:Protein S10 is a component of the 30S ribosomal subunit and participates together with NusB protein in processive transcription Antitermination. The molecular mechanisms by which S10 can act as a translation or a transcription factor are not understood. We used complementation assays and recombineering to delineate regions of S10 dispensable for Antitermination, and determined the crystal structure of a transcriptionally active NusB-S10 complex. In this complex, S10 adopts the same fold as in the 30S subunit and is blocked from simultaneous association with the ribosome. Mass spectrometric mapping of UV-induced crosslinks revealed that the NusB-S10 complex presents an intermolecular, composite, and contiguous binding surface for RNAs containing BoxA Antitermination signals. Furthermore, S10 overproduction complemented a nusB null phenotype. These data demonstrate that S10 and NusB together form a BoxA-binding module, that NusB facilitates entry of S10 into the transcription machinery, and that S10 represents a central hub in processive Antitermination.
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essentiality of ribosomal and transcription Antitermination proteins analyzed by systematic gene replacement in escherichia coli
Journal of Bacteriology, 2007Co-Authors: Mikhail Bubunenko, Teresa Baker, Donald L CourtAbstract:We describe here details of the method we used to identify and distinguish essential from nonessential genes on the bacterial Escherichia coli chromosome. Three key features characterize our method: high-efficiency recombination, precise replacement of just the open reading frame of a chromosomal gene, and the presence of naturally occurring duplications within the bacterial genome. We targeted genes encoding functions critical for processes of transcription and translation. Proteins from three complexes were evaluated to determine if they were essential to the cell by deleting their individual genes. The transcription elongation Nus proteins and termination factor Rho, which are involved in rRNA Antitermination, the ribosomal proteins of the small 30S ribosome subunit, and minor ribosome-associated proteins were analyzed. It was concluded that four of the five bacterial transcription Antitermination proteins are essential, while all four of the minor ribosome-associated proteins examined (RMF, SRA, YfiA, and YhbH), unlike most ribosomal proteins, are dispensable. Interestingly, although most 30S ribosomal proteins were essential, the knockouts of six ribosomal protein genes, rpsF (S6), rpsI (S9), rpsM (S13), rpsO (S15), rpsQ (S17), and rpsT (S20), were viable.
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requirement for nusg for transcription Antitermination in vivo by the λ n protein
Journal of Bacteriology, 2002Co-Authors: Ying Zhou, Donald L Court, Joshua J Filter, Max E Gottesman, David I FriedmanAbstract:Transcription Antitermination by the bacteriophage λ N protein is stimulated in vitro by the Escherichia coli NusG protein. Earlier work suggested that NusG was not required for N activity in vivo. Here we present evidence that NusG also stimulates N-mediated transcription Antitermination in intact cells.
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The alpha subunit of RNA polymerase and transcription Antitermination
Molecular Microbiology, 1996Co-Authors: A. T. Schauer, Donald L Court, Sheau Wei C. Cheng, Chuanhai Zheng, Linda St. Pierre, Diane M. Alessi, Debra L. Hidayetoglu, Nina Costantino, David I FriedmanAbstract:Summary The N gene product of coliphage , with a number of host proteins (Nus factors), regulates phage gene expression by modifying RNA polymerase to a form that overrides transcription-termination signals. Mutations in host nus genes diminish this N-mediated Antitermination. Here, we report the isolation and characterization of the rpoAD305E mutation, a single amino acid change in the carboxy terminal domain (CTD) of the subunit of RNA polymerase, that enhances N-mediated Antitermination. A deletion of the 3 terminus of rpoA, resulting in the expression of an subunit missing the CTD, also enhances Nmediated Antitermination and, similar to rpoAD305E, suppresses the effect of nus mutations. Thus, the N‐ Nus complex may be affected through contacts with the CTD of the subunit of RNA polymerase, as is a group of regulatory proteins that influences initiation of transcription. What distinguishes our findings on the N‐Nus complex from those of previous studies with transcription proteins is that all of the regulators characterized in those studies bind DNA and influence transcription initiation; whereas the N‐Nus complex binds RNA and affects transcription elongation. A screen of some previously identified rpoA mutations that influence transcription activators revealed only one other amino acid change, L290H, in the CTD of the subunit, that influences Antitermination. Although our results provide evidence that interactions of the subunit of RNA polymerase must be considered in forming models of transcription Antitermination, they do not provide information as to whether the interactions of that ultimately influence Antitermination occur during initiation or during elongation of transcription.
Veronique Arluison - One of the best experts on this subject based on the ideXlab platform.
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the sm like rna chaperone hfq mediates transcription Antitermination at rho dependent terminators
The EMBO Journal, 2011Co-Authors: Makhlouf Rabhi, Olivier Espeli, Annie Schwartz, Bastien Cayrol, Rachid A Rahmouni, Veronique ArluisonAbstract:In Escherichia coli, the essential motor protein Rho promotes transcription termination in a tightly controlled manner that is not fully understood. Here, we show that the general post-transcriptional regulatory protein Hfq associates with Rho to regulate Rho function. The Hfq:Rho complex can be further stabilized by RNA bridging both factors in a configuration that inhibits the ATP hydrolysis and duplex unwinding activities of Rho and that mediates transcription Antitermination at Rho-dependent terminators in vitro and in vivo. Antitermination at a prototypical terminator (λtR1) requires Hfq binding to an A/U-rich transcript region directly upstream from the terminator. Antitermination is modulated by trans-acting factors (NusG or nucleic acid competitors) that affect Hfq association with Rho or RNA. These data unveil a new Hfq function and a novel transcription regulatory mechanism with potentially important implications for bacterial RNA metabolism, gene silencing, and pathogenicity.
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The Sm‐like RNA chaperone Hfq mediates transcription Antitermination at Rho‐dependent terminators
The EMBO Journal, 2011Co-Authors: Makhlouf Rabhi, Olivier Espeli, Annie Schwartz, Bastien Cayrol, Veronique Arluison, A. Rachid Rahmouni, Marc BoudvillainAbstract:In Escherichia coli, the essential motor protein Rho promotes transcription termination in a tightly controlled manner that is not fully understood. Here, we show that the general post-transcriptional regulatory protein Hfq associates with Rho to regulate Rho function. The Hfq:Rho complex can be further stabilized by RNA bridging both factors in a configuration that inhibits the ATP hydrolysis and duplex unwinding activities of Rho and that mediates transcription Antitermination at Rho-dependent terminators in vitro and in vivo. Antitermination at a prototypical terminator (λtR1) requires Hfq binding to an A/U-rich transcript region directly upstream from the terminator. Antitermination is modulated by trans-acting factors (NusG or nucleic acid competitors) that affect Hfq association with Rho or RNA. These data unveil a new Hfq function and a novel transcription regulatory mechanism with potentially important implications for bacterial RNA metabolism, gene silencing, and pathogenicity.
Frank J Grundy - One of the best experts on this subject based on the ideXlab platform.
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non conserved residues in clostridium acetobutylicum trnaala contribute to trna tuning for efficient Antitermination of the alas t box riboswitch
Life, 2015Co-Authors: Frank J Grundy, Tina M HenkinAbstract:The T box riboswitch regulates expression of amino acid-related genes in Gram-positive bacteria by monitoring the aminoacylation status of a specific tRNA, the binding of which affects the folding of the riboswitch into mutually exclusive terminator or antiterminator structures. Two main pairing interactions between the tRNA and the leader RNA have been demonstrated to be necessary, but not sufficient, for efficient Antitermination. In this study, we used the Clostridium acetobutylicum alaS gene, which encodes alanyl-tRNA synthetase, to investigate the specificity of the tRNA response. We show that the homologous C. acetobutylicum tRNAAla directs Antitermination of the C. acetobutylicum alaS gene in vitro, but the heterologous Bacillus subtilis tRNAAla (with the same anticodon and acceptor end) does not. Base substitutions at positions that vary between these two tRNAs revealed synergistic and antagonistic effects. Variation occurs primarily at positions that are not conserved in tRNAAla species, which indicates that these non-conserved residues contribute to optimal Antitermination of the homologous alaS gene. This study suggests that elements in tRNAAla may have coevolved with the homologous alaS T box leader RNA for efficient Antitermination.
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kinetic analysis of trna directed transcription Antitermination of the bacillus subtilis glyqs gene in vitro
Journal of Bacteriology, 2004Co-Authors: Frank J Grundy, Tina M HenkinAbstract:Binding of uncharged tRNA to the nascent transcript promotes readthrough of a leader region transcription termination signal in genes regulated by the T box transcription Antitermination mechanism. Each gene in the T box family responds independently to its cognate tRNA, with specificity determined by base pairing of the tRNA to the leader at the anticodon and acceptor ends of the tRNA. tRNA binding stabilizes an antiterminator element in the transcript that sequesters sequences that participate in formation of the terminator helix. tRNAGly-dependent Antitermination of the Bacillus subtilis glyQS leader was previously demonstrated in a purified in vitro assay system. This assay system was used to investigate the kinetics of transcription through the glyQS leader and the effect of tRNA and transcription elongation factors NusA and NusG on transcriptional pausing and Antitermination. Several pause sites, including a major site in the loop of stem III of the leader, were identified, and the effect of modulation of pausing on Antitermination efficiency was analyzed. We found that addition of tRNAGly can promote Antitermination as long as the tRNA is added before the majority of the transcription complexes reach the termination site, and variations in pausing affect the requirements for timing of tRNA addition.
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trna requirements for glyqs Antitermination a new twist on trna
RNA, 2003Co-Authors: Mary R Yousef, Frank J Grundy, Tina M HenkinAbstract:Transcription Antitermination of the Bacillus subtilis glyQS gene, a member of the T box gene regulation family, can be induced during in vitro transcription in a minimal system using purified B. subtilis RNA polymerase by the addition of unmodified T7 RNA polymerase-transcribed tRNAGly. Antitermination was previously shown to depend on base-pairing between the glyQS leader and the tRNA at the anticodon and acceptor ends. In this study, variants of tRNAGly were generated to identify additional tRNA elements required for Antitermination activity, and to determine the effect of structural changes in the tRNA. We find that additions to the 3′ end of the tRNA blocked Antitermination, in agreement with the prediction that uncharged tRNA is the effector in vivo, whereas insertion of 1 nucleotide between the acceptor stem and the 3′ UCCA residues had no effect. Disruption of the D-loop/T-loop tertiary interaction inhibited Antitermination function, as was previously demonstrated for tRNATyr-directed Antitermination of the B. subtilis tyrS gene in vivo. Insertion of a single base pair in the anticodon stem was tolerated, whereas further insertions abolished Antitermination. However, we find that major alterations in the length of the acceptor stem are tolerated, and the insertions exhibited a pattern of periodicity suggesting that there is face-of-the-helix dependence in the positioning of the unpaired UCCA residues at the 3′ end of the tRNA for interaction with the antiterminator bulge and Antitermination.
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trna mediated transcription Antitermination in vitro codon anticodon pairing independent of the ribosome
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Frank J Grundy, Wade C Winkler, Tina M HenkinAbstract:Uncharged tRNA acts as the effector for transcription Antitermination of genes in the T box family in Bacillus subtilis and other Gram-positive bacteria. Genetic studies suggested that expression of these genes is induced by stabilization of an antiterminator element in the leader RNA of each target gene by the cognate uncharged tRNA. The specificity of the tRNA response is dependent on a single codon in the leader, which was postulated to pair with the anticodon of the corresponding tRNA. It was not known whether the leader RNA–tRNA interaction requires additional factors. We show here that tRNA-dependent Antitermination occurs in vitro in a purified transcription system, in the absence of ribosomes or accessory factors, demonstrating that the RNA–RNA interaction is sufficient to control gene expression by Antitermination. The tRNA response exhibits similar specificity in vivo and in vitro, and the Antitermination reaction in vitro is independent of NusA and functions with either B. subtilis or Escherichia coli RNA polymerase.
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analysis of cis acting sequence and structural elements required for Antitermination of the bacillus subtilis tyrs gene
Molecular Microbiology, 1997Co-Authors: Sean M Rollins, Frank J Grundy, Tina M HenkinAbstract:Summary The Bacillus subtilis tyrS gene belongs to the T box family of aminoacyl-tRNA synthetase and amino acid biosynthesis genes, which are regulated by a common mechanism of transcriptional Antitermination. Each gene is induced by specific amino acid limitation; the uncharged cognate tRNA is the effector inducing transcription of the full-length message. The leader regions of the genes in this family share a number of conserved primary sequence and secondary structural elements, the functions of which are unknown. In this study, we examine these regions and report the effects of mutations in several of these elements. In addition, two alternative basepairings in the F box region were found to be necessary for tyrS Antitermination.
