{"database":"biostudies-literature","file_versions":[],"scores":null,"additional":{"submitter":["Jayasinghe OT"],"funding":["HHS | National Institutes of Health","HHS | National Institutes of Health (NIH)","Intramural Research Program","NIGMS NIH HHS"],"pagination":["e0053421"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/S-EPMC9112975"],"repository":["biostudies-literature"],"omics_type":["Unknown"],"volume":["204(5)"],"pubmed_abstract":["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 <i>in vitro</i>. The genome-wide role of such factors in pausing <i>in vivo</i> 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 <i>in vitro</i>. Here, we present the first <i>in vivo</i> 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 <i>in vivo</i>. Based on our thresholding criteria on <i>in vivo</i> 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 <i>in vitro</i> at some of the pause sites identified <i>in vivo</i>. <b>IMPORTANCE</b> NusA is an essential transcription elongation factor that was assumed to play a major role in RNAP pausing. NusA stimulates pausing <i>in vitro</i>; however, the essential nature of NusA had prevented an assessment of its role in pausing <i>in vivo</i>. 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 <i>in vitro</i>. However, NusA did not always stimulate pausing at sites identified <i>in vivo</i>, while in other instances NusA stimulated pausing at sites not observed <i>in vivo</i>. We found that NusA has only a minor role in stimulating RNAP pausing in B. subtilis."],"journal":["Journal of bacteriology"],"pubmed_title":["Transcriptome-Wide Effects of NusA on RNA Polymerase Pausing in Bacillus subtilis."],"pmcid":["PMC9112975"],"funding_grant_id":["GM098399","R01 GM098399"],"pubmed_authors":["Mandell ZF","Jayasinghe OT","Kashlev M","Yakhnin AV","Babitzke P"],"additional_accession":[]},"is_claimable":false,"name":"Transcriptome-Wide Effects of NusA on RNA Polymerase Pausing in Bacillus subtilis.","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 <i>in vitro</i>. The genome-wide role of such factors in pausing <i>in vivo</i> 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 <i>in vitro</i>. Here, we present the first <i>in vivo</i> 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 <i>in vivo</i>. Based on our thresholding criteria on <i>in vivo</i> 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 <i>in vitro</i> at some of the pause sites identified <i>in vivo</i>. <b>IMPORTANCE</b> NusA is an essential transcription elongation factor that was assumed to play a major role in RNAP pausing. NusA stimulates pausing <i>in vitro</i>; however, the essential nature of NusA had prevented an assessment of its role in pausing <i>in vivo</i>. 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 <i>in vitro</i>. However, NusA did not always stimulate pausing at sites identified <i>in vivo</i>, while in other instances NusA stimulated pausing at sites not observed <i>in vivo</i>. We found that NusA has only a minor role in stimulating RNAP pausing in B. subtilis.","dates":{"release":"2022-01-01T00:00:00Z","publication":"2022 May","modification":"2026-06-15T06:06:53.498Z","creation":"2025-04-04T08:01:21.39Z"},"accession":"S-EPMC9112975","cross_references":{"pubmed":["35258320"],"doi":["10.1128/jb.00534-21"]}}