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DksA Guards Elongating RNA Polymerase Against Ribosome-Stalling-Induced Arrest

ABSTRACT: In bacteria, translation-transcription coupling inhibits RNA polymerase (RNAP) stalling. We present evidence suggesting that, upon amino acid starvation, inactive ribosomes promote rather than inhibit RNAP stalling. We developed an algorithm to evaluate genome-wide polymerase progression independently of local noise, and used it to reveal that the transcription factor DksA inhibits promoter-proximal pausing and increases RNAP elongation when uncoupled from translation by depletion of charged tRNAs. DksA has minimal effect on RNAP elongation in vitro and on untranslated RNAs in vivo. In these cases, transcripts can form RNA structures that prevent backtracking. Thus, the effect of DksA on transcript elongation may occur primarily upon ribosome slowing/stalling or at promoter-proximal locations that limit the potential for RNA structure. We propose that inactive ribosomes prevent formation of backtrackblocking mRNA structures and that, in this circumstance, DksA acts as a transcription elongation factor in vivo.

ORGANISM(S): Escherichia coli Escherichia coli str. K-12 substr. MG1655

PROVIDER: GSE54669 | GEO | 2014/04/21

SECONDARY ACCESSION(S): PRJNA237329

REPOSITORIES: GEO

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DksA Guards Elongating RNA Polymerase Against Ribosome-Stalling-Induced Arrest

Project description:In bacteria, translation-transcription coupling inhibits RNA polymerase (RNAP) stalling. We present evidence suggesting that, upon amino acid starvation, inactive ribosomes promote rather than inhibit RNAP stalling. We developed an algorithm to evaluate genome-wide polymerase progression independently of local noise, and used it to reveal that the transcription factor DksA inhibits promoter-proximal pausing and increases RNAP elongation when uncoupled from translation by depletion of charged tRNAs. DksA has minimal effect on RNAP elongation in vitro and on untranslated RNAs in vivo. In these cases, transcripts can form RNA structures that prevent backtracking. Thus, the effect of DksA on transcript elongation may occur primarily upon ribosome slowing/stalling or at promoter-proximal locations that limit the potential for RNA structure. We propose that inactive ribosomes prevent formation of backtrackblocking mRNA structures and that, in this circumstance, DksA acts as a transcription elongation factor in vivo. Chromatin immunoprecipitation (ChIP) experiments were performed by using antibodies against RNA polymerase b subunit in wild-type and DdksA cells treated with 0.5mg/ml serine hydroxamate (SHX) or untreated. DksA and s70 enrichments were compared to RNAP enrichment by ChIP experiments using antibodies against s70 and DksA in wild-type cells (also in DdksA cells as a negative control for DksA ChIP-chip). 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 19 datasets.

2014-04-21 | E-GEOD-54669 | biostudies-arrayexpress

Disributions of RpoC SigA NusA and GreA in Bacillus subtilis

Project description:Bacterial Gre factors associate with RNA polymerase (RNAP) and stimulate intrinsic cleavage of the nascent transcript at the active site of RNAP. Biochemical and genetic studies to date have shown that E. coli Gre factors prevent transcriptional arrest during elongation and enhance transcription fidelity. Furthermore, Gre factors participate in stimulation of promoter escape and suppression of promoter-proximal pausing during beginning of RNA synthesis in E. coli. Although Gre factors are conserved in general bacteria, limited functional studies have been performed in bacteria other than E. coli. In this investigation, ChAP-chip analysis was conducted to visualize the distribution of B. subtilis GreA on the chromosome and determine the effects of GreA inactivation on core RNAP trafficking. Our data show that GreA is uniformly distributed in the transcribed region from the promoter to coding region with core RNAP, and its inactivation induces RNAP accumulation at many promoter or promoter-proximal regions. Based on these findings, we propose that GreA would constantly associate with core RNAP during transcriptional initiation and elongation, and resolves its stalling at promoter or promoter-proximal regions, thus contributing to the even distribution of RNAP along the promoter and coding regions in B. subtilis cells.

2011-04-10 | E-MEXP-3056 | biostudies-arrayexpress

Genome-wide transcriptional profiling of Bacillus subtilis greA deletion and GreA-D44A mutant strains

Project description:Bacterial Gre factors associate with RNA polymerase (RNAP) and stimulate intrinsic cleavage of the nascent transcript at the active site of RNAP. Biochemical and genetic studies to date have shown that E. coli Gre factors prevent transcriptional arrest during elongation and enhance transcription fidelity. Furthermore, Gre factors participate in stimulation of promoter escape and suppression of promoter-proximal pausing during beginning of RNA synthesis in E. coli. Although Gre factors are conserved in general bacteria, limited functional studies have been performed in bacteria other than E. coli. In this investigation, ChAP-chip analysis was conducted to visualize the distribution of B. subtilis GreA on the chromosome and determine the effects of GreA inactivation on core RNAP trafficking. Our data show that GreA inactivation induces RNAP accumulation at many promoter or promoter-proximal regions. Additionally, we performed transcriptome comparison between in wild type and greA deletion and greA D44A mutant cells to see the effect of RNAP pausing to the transcriptomes. Our results indicate that inactivation of GreA has a limited impact on the transcriptome, and these effects are not directly related to RNAP accumulation in the promoter or promoter-proximal regions.

2011-04-10 | E-MEXP-3055 | biostudies-arrayexpress

Rapid transcription of arc/arg3.1 and other very fast immediate early genes in response to neuronal activity is mediated by a stalled RNA polymerase II mechanism

Project description:Transcription of immediate early genes (IEGs) in neurons is exquisitely sensitive to neuronal activity, but the mechanism underlying the earliest of these transcription events is largely unknown. Here we demonstrate that very fast IEGs (VF-IEGs) such as arc/arg3.1 are poised for rapid transcription by the stalling of RNA Polymerase II (Pol II) just downstream of the transcription start site. RNAi-depletion of two subunits of Negative Elongation Factor, a mediator of Pol II stalling, reduces the Pol II occupancy of the arc promoter and compromises the rapid induction of arc and other VF-IEGs. In contrast, reduction of Pol II stalling did not prevent expression of fast IEGs (F-IEGs). These F-IEGs are expressed with comparatively slower kinetics and largely lack promoter proximal Pol II stalling. Taken together, our data strongly indicate that very fast kinetics of neuronal IEG expression require poised Pol II and suggest a role for this mechanism in transcription-dependent learning and memory. Examination of Pol II bindnig genome-wide in rat neurons

2011-05-24 | E-GEOD-22878 | biostudies-arrayexpress

Chromatin Affinity Precipitation-chiop of B. subtilis to gain insight into the invivo dynamics of RNA polymerase

Project description:To obtain an insight into the in vivo dynamics of RNA polymerase (RNAP) on the B. subtilis genome, we analyzed the distribution of ?A and ? subunits of RNAP and the NusA elongation factor on the genome in exponentially growing cells, using the ChAP (Chromatin Affinity Precipitation)-chip method. In contrast to E. coli RNAP, which often accumulates at the promoter-proximal region, B. subtilis RNAP is evenly distributed from the promoter to the coding sequences in the majority of genes. This finding suggests that B. subtilis RNAP recruited to the promoter promptly translocates away from the promoter to form the elongation complex. We detected RNAP accumulation in the promoter-proximal regions of some genes, most of which are attributed to transcription attenuation systems in the leader region. Our findings suggest that the differences in RNAP behavior during initiation and early elongation steps between E. coli and B. subtilis result in distinct strategies for post-initiation control of transcription. The E. coli mechanism involves trapping at the promoter and promoter-proximal pausing of RNAP in addition to transcription attenuation, whereas transcription attenuation in leader sequences is mainly employed in B. subtilis. Wild-type strain, Bacillus subtilis 168, was also used for RNA and genomic DNA extraction and analysis - RNA data was divided by genome DNA data to normalize (1) PCR bias and (2) copy number of RNA molecule per genome for multi-copy genome of exponentially growing bacteria.

2010-06-29 | E-MEXP-2649 | biostudies-arrayexpress

Sensing of individual stalled 80S ribosomes by Fap1 for non-functional rRNA turnover. Li et al

Project description:Cells can respond to stalled ribosomes by sensing ribosome collisions and employing quality control pathways. How ribosome stalling is resolved without collisions, however, has remained elusive. Here, focusing on non-colliding stalling exhibited by decoding-defective ribosomes, we identified Fap1 as a stalling sensor triggering 18S non-functional rRNA decay via poly-ubiquitination of uS3. Ribosome profiling revealed an enrichment of Fap1 at the translation initiation site but also association with elongating individual ribosomes. Cryo-EM structures of Fap1-bound ribosomes elucidated Fap1 probing the mRNA simultaneously at both the entry and exit channels suggesting a mRNA stasis sensing activity, and Fap1 sterically hinders formation of canonical collided di-ribosomes. Our findings indicate that individual stalled ribosomes are the potential signal for ribosome dysfunction, leading to accelerated turnover of the ribosome itself.

2022-09-15 | GSE159329 | GEO

Neurotoxin tetrodotoxin TTX withdrawal induced neuronal activity: RNA Pol II is poised for rapid induction of arc and other neuronal VF-IEGs

Project description:Transcription of immediate early genes (IEGs) in neurons is exquisitely sensitive to neuronal activity, but the mechanism underlying the earliest of these transcription events is largely unknown. Here we demonstrate that very fast IEGs (VF-IEGs) such as arc/arg3.1 are poised for rapid transcription by the stalling of RNA Polymerase II (Pol II) just downstream of the transcription start site. RNAi-depletion of two subunits of a mediator of Pol II stalling, Negative Elongation Factor, reduces Pol II occupancy of the arc promoter and compromises rapid induction of arc and other VF-IEGs. In contrast, reduction of Pol II stalling did not prevent expression of other fast IEGs (F-IEGs). These F-IEGs are expressed with comparatively slower kinetics and largely lack promoter proximal Pol II stalling. Taken together, our data strongly indicate that very fast kinetics of neuronal IEG expression require poised Pol II and suggest a role for this mechanism in transcription-dependent learning and memory. TTX withdrawal induced neuronal activity. To study activity-induced gene expression, neurons were treated with TTX for 48 hours and then TTX was washed out either for 15 minutes (W15) or for 45 minutes (W45). Gene expression was measured in these two groups in comparison to TTX treated neurons.

2011-04-14 | E-GEOD-22622 | biostudies-arrayexpress

Neurotoxin tetrodotoxin TTX withdrawal induced neuronal activity: RNA Pol II is poised for rapid induction of arc and other neuronal VF-IEGs

Project description:Transcription of immediate early genes (IEGs) in neurons is exquisitely sensitive to neuronal activity, but the mechanism underlying the earliest of these transcription events is largely unknown. Here we demonstrate that very fast IEGs (VF-IEGs) such as arc/arg3.1 are poised for rapid transcription by the stalling of RNA Polymerase II (Pol II) just downstream of the transcription start site. RNAi-depletion of two subunits of a mediator of Pol II stalling, Negative Elongation Factor, reduces Pol II occupancy of the arc promoter and compromises rapid induction of arc and other VF-IEGs. In contrast, reduction of Pol II stalling did not prevent expression of other fast IEGs (F-IEGs). These F-IEGs are expressed with comparatively slower kinetics and largely lack promoter proximal Pol II stalling. Taken together, our data strongly indicate that very fast kinetics of neuronal IEG expression require poised Pol II and suggest a role for this mechanism in transcription-dependent learning and memory.

2011-04-15 | GSE22622 | GEO

Rapid transcription of arc/arg3.1 and other very fast immediate early genes in response to neuronal activity is mediated by a stalled RNA polymerase II mechanism

2011-05-24 | GSE22878 | GEO

Distribution of transcription elongation complex in the PTPRN2 gene

Project description:Analysis of RNA polymerase II (RNAP II) binding data in HCT116-C52-tTA cells provides insight into the stalling site of RNAP II in the human PTPRN2 gene.

2018-02-12 | GSE35960 | GEO

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