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

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 s70-dependent transcription pauses in Escherichia coli. Retention of s70 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 s70. 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 s70 and Gre proteins regulate transcription in response to changing environmental cues. Overall design: To identify the genome-wide s70-dependent pause sites, RNET-seq were performed using the E. coli strains s70-WT, s70-greAB-, ß'-WT and ß'-greAB- with His-tag fusion to RpoD (s70) and RpoC (ß') in parallel. The RNAP-DNA-RNA ternary complex immobilized by Ni-NTA agarose beads was treated by DNase I and RNase I to obtain the RNA footprints protected by RNAP. RNA-seq were performed using the same strains and conditions to monitor the changes of gene transcription.