Transcriptome-Wide Effects of NusA on RNA Polymerase Pausing in Bacillus subtilis

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 . The genome-wide role of such factors in pausing 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 . Here, we present the first 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 . Based on our thresholding criteria on 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 at some of the pause sites identified . NusA is an essential transcription elongation factor that was assumed to play a major role in RNAP pausing. NusA stimulates pausing ; however, the essential nature of NusA had prevented an assessment of its role in pausing . 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 . However, NusA did not always stimulate pausing at sites identified , while in other instances NusA stimulated pausing at sites not observed . We found that NusA has only a minor role in stimulating RNAP pausing in B. subtilis.