Nascent RNA sequencing identifies a widespread sigma70-dependent pausing regulated by Gre factors in bacteria

Abstract

Promoter-proximal pausing regulates eukaryotic gene expression and serves as checkpoints to assemble elongation/splicing machinery. Little is known how broadly this type of pausing regulates transcription in bacteria. We apply nascent elongating transcript sequencing combined with RNase I footprinting for genome-wide analysis of σ70-dependent transcription pauses in Escherichia coli. Retention of σ70 induces strong backtracked pauses at a 10−20-bp distance from many promoters. The pauses in the 10−15-bp register of the promoter are dictated by the canonical −10 element, 6−7 nt spacer and “YR+1Y” motif centered at the transcription start site. The promoters for the pauses in the 16−20-bp register contain an additional −10-like sequence recognized by σ70. Our in vitro analysis reveals that DNA scrunching is involved in these pauses relieved by Gre cleavage factors. The genes coding for transcription factors are enriched in these pauses, suggesting that σ70 and Gre proteins regulate transcription in response to changing environmental cues.

Gre Factors Are Required for Biofilm Formation in Salmonella enterica Serovar Typhimurium by Targeting Transcription of the csgD Gene

“…When transcription of the csgD +147 ::lacZ was assessed, a substantial downregulation of lacZ activity in the absence of the Gre factors was detected indicating that the 5′-UTR of csgD is subject to regulation by greA / greB ( Figure 4 B). Thus, we can speculate that Gre factors relieve transcriptional pause(s) in the 5′-untranslated region (5′-UTR) of the csgD gene, a mechanism of action occurring in most cases in 5′-UTR regions as described in the literature [ 3 ].…”

“…In this report, we describe the Gre factors as new players in the array of regulators affecting csgD expression. A potential role for the Gre factors in bacterial adaptation to different environmental stresses has been proposed [ 3 , 7 ]. Hence, expression of greA and greB occurs under different environmental conditions in E. coli [ 3 ].…”

“…A potential role for the Gre factors in bacterial adaptation to different environmental stresses has been proposed [ 3 , 7 ]. Hence, expression of greA and greB occurs under different environmental conditions in E. coli [ 3 ]. The expression of the greA gene is σE-dependent in E. coli and S. Typhimurium, is induced under hypoxia in Mycobacterium avium and under acid stress in Streptococcus mutans [ 34 , 35 , 36 , 37 , 38 ].…”

“…The ubiquitous transcription elongation factors GreA and GreB, henceforth jointly named Gre factors, small cytoplasmic proteins of <20 kDa, have been extensively studied in Escherichia coli . Several roles have been attributed to those proteins: (i) stimulating transcription initiation by likely facilitation of promoter escape, (ii) promoting release of stalling RNA polymerases during transcription elongation by inducing the intrinsic endoribonucleolytic activity of the RNA polymerases, and (iii) chaperone activity promoting refolding of denatured proteins [ 1 , 2 , 3 ]. In E. coli we have previously described that the Gre factors affect expression of biofilm promoting factors such as type 1 fimbriae and flagella [ 4 , 5 ].…”

“…Of note, a more recent report systematically characterized the promoter proximal pauses occurring during transcription in E. coli . It has been suggested that transcriptional pausing represents a widespread regulatory mechanism aimed at modulation of transcriptional output from specific operons in response to changing environmental conditions in a Gre factor dependent manner [ 3 ].…”