Transcriptome-wide effects of NusA on RNA polymerase pausing in Bacillus subtilis
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ABSTRACT:
PROVIDER: PRJNA773124 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR16506711.fastq.gz | Fastqsanger.gz | |||
| SRR16506712.fastq.gz | Fastqsanger.gz | |||
| SRR16506713.fastq.gz | Fastqsanger.gz | |||
| SRR16506714.fastq.gz | Fastqsanger.gz | |||
| SRR16506715.fastq.gz | Fastqsanger.gz |
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Similar Datasets
Project description:In this work we conducted RNET-seq on Wild Type, NusA depletion, nusG deletion, and NusA depletion nusG deletion strains of B. subtilis. Using this data we analysed the transcriptome-wide effects of NusA on RNA polymerase pausing and found that NusA has modest pause stimulating as well as suppressing effects throughout the genome.
2021-10-22 | GSE186285 | GEO
Project description:Transcription elongation is a highly processive process that is punctuated by RNA polymerase (RNAP) pausing. Long-lived pauses can provide time for diverse regulatory events to occur, which play important roles in modulating gene expression. Transcription elongation factors can dramatically affect RNAP pausing in vitro. The genome-wide role of such factors in pausing in vivo has been examined only for NusG in Bacillus subtilis. NusA is another transcription elongation factor known to stimulate pausing of B. subtilis and Escherichia coli RNAP in vitro. Here, we present the first in vivo study to identify the genome-wide role of NusA in RNAP pausing. Using native elongation transcript sequencing followed by RNase digestion (RNET-seq), we analyzed factor-dependent RNAP pausing in B. subtilis and found that NusA has a relatively minor role in RNAP pausing compared to NusG. We demonstrate that NusA has both stimulating and suppressing effects on pausing in vivo. Based on our thresholding criteria on in vivo data, NusA stimulates pausing at 129 pause peaks in 93 different genes or 5' untranslated regions (5' UTRs). Putative pause hairpins were identified for 87 (67%) of the 129 NusA-stimulated pause peaks, suggesting that RNA hairpins are a common component of NusA-stimulated pause signals. However, a consensus sequence was not identified as a NusA-stimulated pause motif. We further demonstrate that NusA stimulates pausing in vitro at some of the pause sites identified in vivo. IMPORTANCE NusA is an essential transcription elongation factor that was assumed to play a major role in RNAP pausing. NusA stimulates pausing in vitro; however, the essential nature of NusA had prevented an assessment of its role in pausing in vivo. Using a NusA depletion strain and RNET-seq, we identified a similar number of NusA-stimulated and NusA-suppressed pause peaks throughout the genome. NusA-stimulated pausing was confirmed at several sites in vitro. However, NusA did not always stimulate pausing at sites identified in vivo, while in other instances NusA stimulated pausing at sites not observed in vivo. We found that NusA has only a minor role in stimulating RNAP pausing in B. subtilis.
| S-EPMC9112975 | biostudies-literature
Project description:Canonical bacterial intrinsic terminators do not require additional factors for efficient transcription termination, although the general transcription elongation factor NusA was known to increase the termination efficiency slightly in vitro. We found that the effect of NusA varies widely among different terminators and identified a subclass of weak non-canonical terminators that largely depend on NusA for recognition by RNA polymerase. Using genome-wide 3â end-mapping on an engineered Bacillus subtilis NusA depletion strain, we identified >2000 intrinsic terminators, hundreds of which are NusA-dependent. Our in vitro and in vivo characterization showed that terminators with weak RNA hairpins and distal U-tract interruptions tend to be NusA-dependent. Our observations indicate that the lethality associated with deletion of nusA is caused by global readthrough of NusA-dependent terminators, resulting in misregulation of physiologically important downstream genes. We also show that nusA expression is autoregulated by a transcription attenuation mechanism that is mediated by NusA-dependent termination. 3' end-mapping and mRNA profiling of stationary phase B. subtilis NusA-depletion (PLBS802) strain in NusA expressing and depleted conditions, 6 replicates each.
2016-07-03 | E-GEOD-67058 | biostudies-arrayexpress
Project description:Canonical bacterial intrinsic terminators do not require additional factors for efficient transcription termination, although the general transcription elongation factor NusA was known to increase the termination efficiency slightly in vitro. We found that the effect of NusA varies widely among different terminators and identified a subclass of weak non-canonical terminators that largely depend on NusA for recognition by RNA polymerase. Using genome-wide 3’ end-mapping on an engineered Bacillus subtilis NusA depletion strain, we identified >2000 intrinsic terminators, hundreds of which are NusA-dependent. Our in vitro and in vivo characterization showed that terminators with weak RNA hairpins and distal U-tract interruptions tend to be NusA-dependent. Our observations indicate that the lethality associated with deletion of nusA is caused by global readthrough of NusA-dependent terminators, resulting in misregulation of physiologically important downstream genes. We also show that nusA expression is autoregulated by a transcription attenuation mechanism that is mediated by NusA-dependent termination.
2016-01-01 | GSE67058 | GEO
Project description:To construct a regulatory map of the genome of the human pathogen, Mycobacterium tuberculosis, we applied two complementary high-resolution approaches, strand-specific RNA-seq and ChIP-seq, to survey the global transcriptome and monitor genome-wide dynamics of RNA polymerase (RNAP) and NusA. ChIP-seq revealed that RNAP and the antiterminator, NusA, occurred ubiquitously with the NusA profile mirroring RNAP distribution in both the exponential and stationary phases of growth. Generally, promoter-proximal peaks for RNAP and NusA were observed, followed by a decrease in signal strength reflecting transcriptional polarity. Differential binding of RNAP and NusA in the two growth conditions correlated with transcriptional activity as reflected by RNA abundance. Indeed, a significant association between expression levels and the presence of NusA throughout the gene body was detected, confirming the peculiar transcription-promoting role of NusA. Integration of the datasets pinpointed transcriptional units, mapped promoters and uncovered new anti-sense and non-coding transcripts. Highly expressed transcriptional units were situated mainly on the leading strand, despite the relatively unbiased distribution of genes throughout the genome, thus helping the replicative and transcriptional complexes to align.
2012-12-01 | GSE40846 | GEO
Project description:To construct a regulatory map of the genome of the human pathogen, Mycobacterium tuberculosis, we applied two complementary high-resolution approaches, strand-specific RNA-seq and ChIP-seq, to survey the global transcriptome and monitor genome-wide dynamics of RNA polymerase (RNAP) and NusA. ChIP-seq revealed that RNAP and the antiterminator, NusA, occurred ubiquitously with the NusA profile mirroring RNAP distribution in both the exponential and stationary phases of growth. Generally, promoter-proximal peaks for RNAP and NusA were observed, followed by a decrease in signal strength reflecting transcriptional polarity. Differential binding of RNAP and NusA in the two growth conditions correlated with transcriptional activity as reflected by RNA abundance. Indeed, a significant association between expression levels and the presence of NusA throughout the gene body was detected, confirming the peculiar transcription-promoting role of NusA. Integration of the datasets pinpointed transcriptional units, mapped promoters and uncovered new anti-sense and non-coding transcripts. Highly expressed transcriptional units were situated mainly on the leading strand, despite the relatively unbiased distribution of genes throughout the genome, thus helping the replicative and transcriptional complexes to align.
2012-12-01 | GSE40845 | GEO
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
Project description:To construct a regulatory map of the genome of the human pathogen, Mycobacterium tuberculosis, we applied two complementary high-resolution approaches, strand-specific RNA-seq and ChIP-seq, to survey the global transcriptome and monitor genome-wide dynamics of RNA polymerase (RNAP) and NusA. ChIP-seq revealed that RNAP and the antiterminator, NusA, occurred ubiquitously with the NusA profile mirroring RNAP distribution in both the exponential and stationary phases of growth. Generally, promoter-proximal peaks for RNAP and NusA were observed, followed by a decrease in signal strength reflecting transcriptional polarity. Differential binding of RNAP and NusA in the two growth conditions correlated with transcriptional activity as reflected by RNA abundance. Indeed, a significant association between expression levels and the presence of NusA throughout the gene body was detected, confirming the peculiar transcription-promoting role of NusA. Integration of the datasets pinpointed transcriptional units, mapped promoters and uncovered new anti-sense and non-coding transcripts. Highly expressed transcriptional units were situated mainly on the leading strand, despite the relatively unbiased distribution of genes throughout the genome, thus helping the replicative and transcriptional complexes to align. ChIP-Seq of RNAP and NusA in Mtb H37Rv at exponential and stationary phase cultures. Each experiment was performed in duplicate. Input DNA (No IP) was used as a control.
2012-12-01 | E-GEOD-40845 | biostudies-arrayexpress
Project description:To construct a regulatory map of the genome of the human pathogen, Mycobacterium tuberculosis, we applied two complementary high-resolution approaches, strand-specific RNA-seq and ChIP-seq, to survey the global transcriptome and monitor genome-wide dynamics of RNA polymerase (RNAP) and NusA. ChIP-seq revealed that RNAP and the antiterminator, NusA, occurred ubiquitously with the NusA profile mirroring RNAP distribution in both the exponential and stationary phases of growth. Generally, promoter-proximal peaks for RNAP and NusA were observed, followed by a decrease in signal strength reflecting transcriptional polarity. Differential binding of RNAP and NusA in the two growth conditions correlated with transcriptional activity as reflected by RNA abundance. Indeed, a significant association between expression levels and the presence of NusA throughout the gene body was detected, confirming the peculiar transcription-promoting role of NusA. Integration of the datasets pinpointed transcriptional units, mapped promoters and uncovered new anti-sense and non-coding transcripts. Highly expressed transcriptional units were situated mainly on the leading strand, despite the relatively unbiased distribution of genes throughout the genome, thus helping the replicative and transcriptional complexes to align. RNA-Seq in exponential and stationary phase cultures
2012-12-01 | E-GEOD-40846 | biostudies-arrayexpress
Project description:In this work we conducted Term-seq on Wild Type, NusA depletion, nusG deletion, and NusA depletion nusG deletion strains of B. subtilis. Using this approach, we found that NusG is an intrinsic termination factor that works single and cooperatively with NusA.
2021-04-19 | GSE154522 | GEO