Bacteroides expand the functional versatility of a universal transcription factor and transcribed DNA to program capsule diversity
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ABSTRACT:
ORGANISM(S): Bacteroides fragilis NCTC 9343
PROVIDER: GSE281607 | GEO | 2024/11/11
REPOSITORIES: GEO
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Project description:Accurate translation of mRNAs into functional proteins is a fundamental process in all living organisms. In bacteria, in the early stage of translation elongation, peptidyl-tRNAs (pep-tRNAs) with short nascent chains frequently dissociate from the ribosome (pep-tRNA drop-off). The dissociated pep-tRNAs are deacylated and recycled by peptidyl-tRNA hydrolase (PTH), which is an essential enzyme in bacteria. Here, we establish a highly sensitive method for direct profiling of pep-tRNAs using RNA isolation method and mass spectrometry. We isolated each tRNA species with peptide from Escherichia coli pthts cells using reciprocal circulating chromatography and precisely analyzed their nascent peptides. As a result, we successfully detected 703 peptides consisted of 402 cognate peptides and 301 non-cognate peptides with single amino-acid substitution. Detailed analysis of individual pep-tRNAs revealed that most of the substitutions in the miscoded peptides take place at the C-terminal drop-off site. We further examined this observation using a reporter construct and found that the non-cognate pep-tRNAs produced by mistranslation rarely participate in the next round of elongation but dissociate from the ribosome, suggesting that pep-tRNA drop-off is an active mechanism by which the ribosome rejects miscoded pep-tRNAs in the early elongation, thereby contributing to quality control of protein synthesis after peptide bond formation.
2023-05-18 | PXD030807 | Pride
Project description:Accurate translation of mRNAs into functional proteins is a fundamental process in all living organisms. In bacteria, in the early stage of translation elongation, peptidyl-tRNAs (pep-tRNAs) with short nascent chains frequently dissociate from the ribosome (pep-tRNA drop-off). The dissociated pep-tRNAs are deacylated and recycled by peptidyl-tRNA hydrolase (PTH), which is an essential enzyme in bacteria. Here, we establish a highly sensitive method for direct profiling of pep-tRNAs using RNA isolation method and mass spectrometry. We isolated each tRNA species with peptide from Escherichia coli pthts cells using reciprocal circulating chromatography and precisely analyzed their nascent peptides. As a result, we successfully detected 703 peptides consisted of 402 cognate peptides and 301 non-cognate peptides with single amino-acid substitution. Detailed analysis of individual pep-tRNAs revealed that most of the substitutions in the miscoded peptides take place at the C-terminal drop-off site. We further examined this observation using a reporter construct and found that the non-cognate pep-tRNAs produced by mistranslation rarely participate in the next round of elongation but dissociate from the ribosome, suggesting that pep-tRNA drop-off is an active mechanism by which the ribosome rejects miscoded pep-tRNAs in the early elongation, thereby contributing to quality control of protein synthesis after peptide bond formation.
2023-05-18 | PXD030808 | Pride
Project description:Accurate translation of mRNAs into functional proteins is a fundamental process in all living organisms. In bacteria, in the early stage of translation elongation, peptidyl-tRNAs (pep-tRNAs) with short nascent chains frequently dissociate from the ribosome (pep-tRNA drop-off). The dissociated pep-tRNAs are deacylated and recycled by peptidyl-tRNA hydrolase (PTH), which is an essential enzyme in bacteria. Here, we establish a highly sensitive method for direct profiling of pep-tRNAs using RNA isolation method and mass spectrometry. We isolated each tRNA species with peptide from Escherichia coli pthts cells using reciprocal circulating chromatography and precisely analyzed their nascent peptides. As a result, we successfully detected 703 peptides consisted of 402 cognate peptides and 301 non-cognate peptides with single amino-acid substitution. Detailed analysis of individual pep-tRNAs revealed that most of the substitutions in the miscoded peptides take place at the C-terminal drop-off site. We further examined this observation using a reporter construct and found that the non-cognate pep-tRNAs produced by mistranslation rarely participate in the next round of elongation but dissociate from the ribosome, suggesting that pep-tRNA drop-off is an active mechanism by which the ribosome rejects miscoded pep-tRNAs in the early elongation, thereby contributing to quality control of protein synthesis after peptide bond formation.
2023-05-18 | PXD030806 | Pride
Project description:Accurate translation of mRNAs into functional proteins is a fundamental process in all living organisms. In bacteria, in the early stage of translation elongation, peptidyl-tRNAs (pep-tRNAs) with short nascent chains frequently dissociate from the ribosome (pep-tRNA drop-off). The dissociated pep-tRNAs are deacylated and recycled by peptidyl-tRNA hydrolase (PTH), which is an essential enzyme in bacteria. Here, we establish a highly sensitive method for direct profiling of pep-tRNAs using RNA isolation method and mass spectrometry. We isolated each tRNA species with peptide from Escherichia coli pthts cells using reciprocal circulating chromatography and precisely analyzed their nascent peptides. As a result, we successfully detected 703 peptides consisted of 402 cognate peptides and 301 non-cognate peptides with single amino-acid substitution. Detailed analysis of individual pep-tRNAs revealed that most of the substitutions in the miscoded peptides take place at the C-terminal drop-off site. We further examined this observation using a reporter construct and found that the non-cognate pep-tRNAs produced by mistranslation rarely participate in the next round of elongation but dissociate from the ribosome, suggesting that pep-tRNA drop-off is an active mechanism by which the ribosome rejects miscoded pep-tRNAs in the early elongation, thereby contributing to quality control of protein synthesis after peptide bond formation.
2023-05-18 | PXD030805 | Pride
Project description:The in vivo trafficking patterns on DNA by the bacterial regulators of transcript elongation Sigma70, Rho, NusA, and NusG and the explanation for high promoter-proximal levels or peaks of RNA polymerase (RNAP) are unknown. Genome-wide ChIP-chip on E. coli revealed distinct association patterns of regulators as RNAP transcribes away from promoters (Rho first, then NusA, and then NusG). However, the interactions of elongating complexes with these regulators, including a weak interaction with Sigma70, did not differ significantly among most transcription units. A modest variation of NusG signal among genes reflected increased NusG interaction as transcription progresses, rather than functional specialization of elongating complexes. Promoter-proximal RNAP peaks were offset from Sigma70 peaks in the direction of transcription and co-occurred with NusA and Rho peaks, suggesting that the RNAP peaks reflected elongating, rather than initiating, complexes. However, inhibition of Rho did not increase RNAP levels within genes downstream of the RNAP peaks, suggesting the peaks are caused by a mechanism other than simple Rho-dependent attenuation.
2009-01-03 | GSE13938 | GEO
Project description:The in vivo trafficking patterns on DNA by the bacterial regulators of transcript elongation Sigma70, Rho, NusA, and NusG and the explanation for high promoter-proximal levels or peaks of RNA polymerase (RNAP) are unknown. Genome-wide ChIP-chip on E. coli revealed distinct association patterns of regulators as RNAP transcribes away from promoters (Rho first, then NusA, and then NusG). However, the interactions of elongating complexes with these regulators, including a weak interaction with Sigma70, did not differ significantly among most transcription units. A modest variation of NusG signal among genes reflected increased NusG interaction as transcription progresses, rather than functional specialization of elongating complexes. Promoter-proximal RNAP peaks were offset from Sigma70 peaks in the direction of transcription and co-occurred with NusA and Rho peaks, suggesting that the RNAP peaks reflected elongating, rather than initiating, complexes. However, inhibition of Rho did not increase RNAP levels within genes downstream of the RNAP peaks, suggesting the peaks are caused by a mechanism other than simple Rho-dependent attenuation. Chromatin immunoprecipitation (ChIP) experiments were performed using antibodies against RNA polymerase (Beta' subunit), Sigma70, NusA, NusG, or Rho. Differentially labeled ChIP DNA and genomic DNA were competitively hybridized to an E. coli K-12 MG1655 tiling array with overlapping probes at ~24bp spacing across the entire genome. The series contains 17 total datasets.
2010-05-14 | E-GEOD-13938 | biostudies-arrayexpress
Project description:Despite the prevalence of antisense transcripts in bacterial transcriptomes, little is known about how their synthesis is controlled. We report that a major function of the Escherichia coli termination factor Rho and its co-factor NusG is suppression of ubiquitous antisense transcription genome-wide. Rho binds C-rich unstructured nascent RNA (high C/G ratio) prior to its ATP-dependent dissociation of transcription complexes. NusG is required for efficient termination at minority subsets (~20%) of both antisense and sense Rho-dependent terminators with lower C/G ratio sequences. In contrast, a widely studied nusA deletion proposed to compromise Rho-dependent termination had no effect on antisense or sense Rho-dependent terminators in vivo. Global co-localization of the nucleoid-associated protein H-NS with Rho-dependent terminators and genetic interactions between hns and rho suggest that H-NS aids Rho in suppression of antisense transcription. The combined actions of Rho, NusG, and H-NS appear to be analogous to the Sen1-Nrd1-Nab3 and nucleosome systems that suppress antisense transcription in eukaryotes. Tiling expression microarray experiments were performed in cells treated with 20 ug/ml bicyclomycin or cells deleted for nusG, or a partial deletion of nusA. Labeled cDNAs were hybridized to an E. coli K-12 MG1655 tiling array with overlapping probes at ~12bp spacing across the entire genome. The series contains 14 datasets.
2012-11-01 | E-GEOD-41938 | biostudies-arrayexpress
Project description:Despite the prevalence of antisense transcripts in bacterial transcriptomes, little is known about how their synthesis is controlled. We report that a major function of the Escherichia coli termination factor Rho and its co-factor NusG is suppression of ubiquitous antisense transcription genome-wide. Rho binds C-rich unstructured nascent RNA (high C/G ratio) prior to its ATP-dependent dissociation of transcription complexes. NusG is required for efficient termination at minority subsets (~20%) of both antisense and sense Rho-dependent terminators with lower C/G ratio sequences. In contrast, a widely studied nusA deletion proposed to compromise Rho-dependent termination had no effect on antisense or sense Rho-dependent terminators in vivo. Global co-localization of the nucleoid-associated protein H-NS with Rho-dependent terminators and genetic interactions between hns and rho suggest that H-NS aids Rho in suppression of antisense transcription. The combined actions of Rho, NusG, and H-NS appear to be analogous to the Sen1-Nrd1-Nab3 and nucleosome systems that suppress antisense transcription in eukaryotes. RNA-seq experiments were performed on ribosome-depleted RNA from cells treated with 20 ug/ml bicyclomycin or untreated cells. The series contains 4 datasets.
2012-11-01 | E-GEOD-41939 | biostudies-arrayexpress
Project description:Despite the prevalence of antisense transcripts in bacterial transcriptomes, little is known about how their synthesis is controlled. We report that a major function of the Escherichia coli termination factor Rho and its co-factor NusG is suppression of ubiquitous antisense transcription genome-wide. Rho binds C-rich unstructured nascent RNA (high C/G ratio) prior to its ATP-dependent dissociation of transcription complexes. NusG is required for efficient termination at minority subsets (~20%) of both antisense and sense Rho-dependent terminators with lower C/G ratio sequences. In contrast, a widely studied nusA deletion proposed to compromise Rho-dependent termination had no effect on antisense or sense Rho-dependent terminators in vivo. Global co-localization of the nucleoid-associated protein H-NS with Rho-dependent terminators and genetic interactions between hns and rho suggest that H-NS aids Rho in suppression of antisense transcription. The combined actions of Rho, NusG, and H-NS appear to be analogous to the Sen1-Nrd1-Nab3 and nucleosome systems that suppress antisense transcription in eukaryotes. Chromatin immunoprecipitation (ChIP) experiments were performed using antibodies against RNA polymerase (RNAP; Beta subunit) in wild-type cells or cells deleted for hns, nusG, or a partial deletion of nusA. Differentially labeled ChIP DNA and genomic DNA were competitively hybridized to an E. coli K-12 MG1655 tiling array with overlapping probes at ~12bp spacing across the entire genome. The series contains 12 datasets.
2012-11-01 | E-GEOD-41936 | biostudies-arrayexpress
Project description:Expression of virulence genes in pathogenic E. coli is controlled in part by the transcription silencer H-NS and its paralogs (e.g., StpA), which sequester DNA in multi-kb nucleoprotein filaments to inhibit transcription initiation, elongation, or both. Some activators counter-silence initiation by displacing H-NS from promoters. How H-NS inhibition of elongation is overcome is not understood. In uropathogenic E. coli (UPEC), elongation regulator RfaH aids expression of some H-NS-silenced pathogenicity operons (e.g., hlyCABD encoding hemolysin). RfaH associates with elongation complexes (ECs) via direct contacts to a transiently exposed, nontemplate DNA-strand sequence called ops (operon polarity suppressor). RfaH–ops interactions establish long-lived RfaH–EC contacts that allow RfaH to recruit ribosomes to the nascent mRNA and to suppress transcriptional pausing and termination. Using ChIP-seq, we mapped the genome-scale distributions of RfaH, H-NS, StpA, RNA polymerase (RNAP), and σ70 in the UPEC strain CFT073. We identify 8 RfaH-activated operons, all of which were bound by H-NS and StpA. Four are new additions to the RfaH regulon. Deletion of RfaH caused premature termination whereas deletion of H-NS and StpA allowed elongation without RfaH. Thus, RfaH is an elongation counter-silencer of H-NS. Consistent with elongation counter-silencing, deletion of StpA alone decreased the effect of RfaH. StpA increases DNA bridging, which inhibits transcript elongation via topological constraints on RNAP. Residual RfaH effect when both H-NS and StpA were deleted was attributable to targeting of RfaH-regulated operons by a minor H-NS paralog, Hfp. These operons have evolved higher levels of H-NS–binding features, explaining minor-paralog targeting.
2022-10-06 | GSE212064 | GEO