NusG controls transcription pausing and RNA polymerase translocation throughout the Bacillus subtilis genome

Abstract

Transcription is punctuated by RNA polymerase (RNAP) pausing. These pauses provide time for diverse regulatory events that can modulate gene expression. Transcription elongation factors dramatically affect RNAP pausing in vitro, but the genome-wide role of such factors on pausing has not been examined. Using native elongating transcript sequencing followed by RNase digestion (RNET-seq), we analyzed RNAP pausing in Bacillus subtilis genome-wide and identified an extensive role of NusG in pausing. This universally conserved transcription elongation factor is known as Spt5 in archaeal and eukaryotic organisms. B. subtilis NusG shifts RNAP to the posttranslocation register and induces pausing at 1,600 sites containing a consensus TTNTTT motif in the nontemplate DNA strand within the paused transcription bubble. The TTNTTT motif is necessary but not sufficient for NusG-dependent pausing. Approximately one-fourth of these pause sites were localized to untranslated regions and could participate in posttranscription initiation control of gene expression as was previously shown for tlrB and the trpEDCFBA operon. Most of the remaining pause sites were identified in protein-coding sequences. NusG-dependent pausing was confirmed for all 10 pause sites that we tested in vitro. Putative pause hairpins were identified for 225 of the 342 strongest NusG-dependent pause sites, and some of these hairpins were shown to function in vitro. NusG-dependent pausing in the ribD riboswitch provides time for cotranscriptional binding of flavin mononucleotide, which decreases the concentration required for termination upstream of the ribD coding sequence. Our phylogenetic analysis implicates NusG-dependent pausing as a widespread mechanism in bacteria.

NusG, an Ancient Yet Rapidly Evolving Transcription Factor

“…In E. coli , RNAP pauses frequently and NusG facilitates RNA synthesis ( Herbert et al, 2010 ). By contrast, B. subtilis RNAP rarely pauses and NusG stimulates pausing in vitro and in vivo ( Yakhnin et al, 2020a ). Unlike E. coli NusG, which is positioned next to the non-template DNA strand in the TEC but is not known to recognize any specific DNA elements ( Kang et al, 2018 ), B. subtilis NusG specifically binds to T-rich DNA sequences and delays RNA chain elongation ( Yakhnin et al, 2016 ).…”

“…By contrast, in B. subtilis , NusG recognizes a simpler consensus TTNTTT motif and stimulates pausing genome wide, favoring forward translocation of RNAP ( Yakhnin et al, 2020a ). Sequences that induce intrinsic, NusG-independent pausing of B. subtilis enzyme are also very different from the consensus pause elements documented in E. coli , and backtracking is not observed ( Yakhnin et al, 2020a ). Although the mechanism and regulation of pausing appear to be distinct, slowing RNAP is expected to be essential in both B. subtilis and E. coli .…”

“…Pausing-defective E. coli RNAP variants do not support cell growth but can be rescued by small-molecule ligands that slow the RNAP down ( Artsimovitch et al, 2003 ). In contrast to E. coli RNAP, which readily pauses at consensus sequences without the aid of accessory factors ( Artsimovitch and Landick, 2000 ; Larson et al, 2014 ; Vvedenskaya et al, 2014 ), B. subtilis RNAP relies on NusG to slow it down ( Yakhnin et al, 2016 , 2020a ). In this light, NusG can be viewed as a pause-promoting accessory subunit, a regulatory mechanism that could be widespread in bacteria ( Yakhnin et al, 2020b ).…”

Requirements for efficient ligand-gated co-transcriptional switching in designed variants of the B. subtilis pbuE adenine-responsive riboswitch in E. coli

“…Many riboswitches contain stretches of uridines in their expression platforms that represent RNA polymerase pause sites that give more time for events such as RNA folding and ligand binding to occur [13,17,54,55]. In the pbuE riboswitch, the P4 hexauridine tract has been observed to be a pause site in vitro using E. coli and B. subtilis RNAP [30], but was not identified as a pause site in two genome-wide surveys of transcriptional pausing in B. subtilis [56,57]. In the design of the P4 length variant series, only P4-8 bp and P4-10 bp retain the hexauridine tract; the other variants reduced or eliminate this tract.…”

Basic mechanisms and kinetics of pause-interspersed transcript elongation

“…In addition, the effects of transcriptional modulators are overlooked in most models, and none address heterogeneity of pausing among species. For example, in E. coli , NusG is an anti-pausing factor that could stimulate forward translocation and prevent RNAP backtracking, while in Bacillus subtilis , NusG induces pausing by shifting RNAP to the post-translocation register ( 26 , 81–84 ). Another example is that E. coli RNAP recognizes a well-characterized hairpin-stabilized his pause site, while B. subtilis RNAP and mammalian Pol II do not respond to this hairpin-mediated signal ( 26 , 85 ).…”