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 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.

Key players in regulatory RNA realm of bacteria.

Precise regulation of gene expression is crucial for living cells to adapt for survival in diverse environmental conditions. Among the common cellular regulatory mechanisms, RNA-based regulators play a key role in all domains of life. Discovery of regulatory RNAs have made a paradigm shift in molecular biology as many regulatory functions of RNA have been identified beyond its canonical roles as messenger, ribosomal and transfer RNA. In the complex regulatory RNA network, riboswitches, small RNAs, and RNA thermometers can be identified as some of the key players. Herein, we review the discovery, mechanism, and potential therapeutic use of these classes of regulatory RNAs mainly found in bacteria. Being highly adaptive organisms that inhabit a broad range of ecological niches, bacteria have adopted tight and rapid-responding gene regulation mechanisms. This review aims to highlight how bacteria utilize versatile RNA structures and sequences to build a sophisticated gene regulation network.