The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Véronique Arluison - One of the best experts on this subject based on the ideXlab platform.
-
the sm like rna chaperone hfq mediates transcription antitermination at rho dependent terminators
The EMBO Journal, 2011Co-Authors: Rachid A Rahmouni, Makhlouf Rabhi, Annie Schwartz, Olivier Espéli, Bastien Cayrol, Véronique 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.
-
the sm like rna chaperone hfq mediates transcription antitermination at rho dependent terminators
The EMBO Journal, 2011Co-Authors: Rachid A Rahmouni, Makhlouf Rabhi, Annie Schwartz, Olivier Espéli, Bastien Cayrol, Véronique 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.
-
The Sm‐like RNA chaperone Hfq mediates transcription antitermination at Rho‐dependent terminators
The EMBO Journal, 2011Co-Authors: Makhlouf Rabhi, A. Rachid Rahmouni, Annie Schwartz, Olivier Espéli, Bastien Cayrol, Véronique Arluison, 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.
Tina M. Henkin - One of the best experts on this subject based on the ideXlab platform.
-
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.
-
codon anticodon recognition in the bacillus subtilis glyqs t box riboswitch rna dependent codon selection outside the ribosome
Journal of Biological Chemistry, 2015Co-Authors: Enrico Caserta, Frank J Grundy, Tina M. HenkinAbstract:Abstract Many amino acid-related genes in Gram-positive bacteria are regulated by the T box riboswitch. The leader RNA of genes in the T box family controls the expression of downstream genes by monitoring the aminoacylation status of the cognate tRNA. Previous studies identified a three-nucleotide codon, termed the “Specifier Sequence,” in the riboswitch that corresponds to the amino acid identity of the downstream genes. Pairing of the Specifier Sequence with the anticodon of the cognate tRNA is the primary determinant of specific tRNA recognition. This interaction mimics codon-anticodon pairing in translation but occurs in the absence of the ribosome. The goal of the current study was to determine the effect of a full range of mismatches for comparison with codon recognition in translation. Mutations were individually introduced into the Specifier Sequence of the glyQS leader RNA and tRNAGly anticodon to test the effect of all possible pairing combinations on tRNA binding affinity and antitermination efficiency. The functional role of the conserved purine 3′ of the Specifier Sequence was also verifiedin this study. We found that substitutions at the Specifier Sequence resulted in reduced binding, the magnitude of which correlates well with the predicted stability of the RNA-RNA pairing. However, the tolerance for specific mismatches in antitermination was generally different from that during decoding, which reveals a unique tRNA recognition pattern in the T box antitermination system.
-
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.
-
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.
-
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.
Makhlouf Rabhi - One of the best experts on this subject based on the ideXlab platform.
-
the sm like rna chaperone hfq mediates transcription antitermination at rho dependent terminators
The EMBO Journal, 2011Co-Authors: Rachid A Rahmouni, Makhlouf Rabhi, Annie Schwartz, Olivier Espéli, Bastien Cayrol, Véronique 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.
-
the sm like rna chaperone hfq mediates transcription antitermination at rho dependent terminators
The EMBO Journal, 2011Co-Authors: Rachid A Rahmouni, Makhlouf Rabhi, Annie Schwartz, Olivier Espéli, Bastien Cayrol, Véronique 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.
-
The Sm‐like RNA chaperone Hfq mediates transcription antitermination at Rho‐dependent terminators
The EMBO Journal, 2011Co-Authors: Makhlouf Rabhi, A. Rachid Rahmouni, Annie Schwartz, Olivier Espéli, Bastien Cayrol, Véronique Arluison, 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.
Bastien Cayrol - One of the best experts on this subject based on the ideXlab platform.
-
the sm like rna chaperone hfq mediates transcription antitermination at rho dependent terminators
The EMBO Journal, 2011Co-Authors: Rachid A Rahmouni, Makhlouf Rabhi, Annie Schwartz, Olivier Espéli, Bastien Cayrol, Véronique 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.
-
the sm like rna chaperone hfq mediates transcription antitermination at rho dependent terminators
The EMBO Journal, 2011Co-Authors: Rachid A Rahmouni, Makhlouf Rabhi, Annie Schwartz, Olivier Espéli, Bastien Cayrol, Véronique 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.
-
The Sm‐like RNA chaperone Hfq mediates transcription antitermination at Rho‐dependent terminators
The EMBO Journal, 2011Co-Authors: Makhlouf Rabhi, A. Rachid Rahmouni, Annie Schwartz, Olivier Espéli, Bastien Cayrol, Véronique Arluison, 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.
-
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.
-
codon anticodon recognition in the bacillus subtilis glyqs t box riboswitch rna dependent codon selection outside the ribosome
Journal of Biological Chemistry, 2015Co-Authors: Enrico Caserta, Frank J Grundy, Tina M. HenkinAbstract:Abstract Many amino acid-related genes in Gram-positive bacteria are regulated by the T box riboswitch. The leader RNA of genes in the T box family controls the expression of downstream genes by monitoring the aminoacylation status of the cognate tRNA. Previous studies identified a three-nucleotide codon, termed the “Specifier Sequence,” in the riboswitch that corresponds to the amino acid identity of the downstream genes. Pairing of the Specifier Sequence with the anticodon of the cognate tRNA is the primary determinant of specific tRNA recognition. This interaction mimics codon-anticodon pairing in translation but occurs in the absence of the ribosome. The goal of the current study was to determine the effect of a full range of mismatches for comparison with codon recognition in translation. Mutations were individually introduced into the Specifier Sequence of the glyQS leader RNA and tRNAGly anticodon to test the effect of all possible pairing combinations on tRNA binding affinity and antitermination efficiency. The functional role of the conserved purine 3′ of the Specifier Sequence was also verifiedin this study. We found that substitutions at the Specifier Sequence resulted in reduced binding, the magnitude of which correlates well with the predicted stability of the RNA-RNA pairing. However, the tolerance for specific mismatches in antitermination was generally different from that during decoding, which reveals a unique tRNA recognition pattern in the T box antitermination system.
-
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.
-
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.
-
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.