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

Promoter-proximal pausing regulates expression of many eukaryotic genes and serves as checkpoints for assembly of elongation/splicing machinery. Little is known how broadly the pausing is employed in transcriptional regulation in bacteria. We applied NET-seq combined with RNase I footprinting for genome-wide analysis of {sigma}70-dependent transcription pauses in Escherichia coli. Many E. coli genes appear to contain clusters of strong backtracked pauses at 10-20-bp distance from the transcription start site caused by retention of {sigma}70 subunit in RNA polymerase. The pauses in 10-15-bp register of the promoter are dictated by binding of {sigma}70 to canonical -10 element, 6-7 nt spacer and "YR+1Y" motif centered at transcription start site all characteristic for strong E. coli promoters. The promoters for the pauses in 16-20-bp register contain an additional -10-like sequence positioned on the same face of the DNA duplex as the original -10 element suggesting that {sigma}70 hopping was responsible for these pauses. Our in vitro analysis reveals that RNA polymerase backtracking and DNA scrunching are involved in these pauses that are relieved by Gre transcript cleavage factors. The genes coding for transcription factors are enriched in these pauses suggesting that {sigma}70 and Gre proteins regulate transcription in response to changing environmental cues.