Prevalence of RNA polymerase stalling at Escherichia coli promoters after open complex formation

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Abstract
RNA polymerase (RNAP) trapped in intermediate stages of promoter escape, as well as RNAP paused at promoter-proximal σ70-dependent pause sites, gives rise to stable, transcriptionally engaged stalled complexes that can limit promoter function and present potential sites for transcription regulation. To investigate the prevalence of such intermediates, we screened 118 Escherichia coli candidate promoters for RNAP stalling at or near the promoter, using in vivo KMnO4 mapping of RNAP on chromosomal DNA. Of 34 active promoters, the seven preceding lacZ, tnaA, cspA, cspD, rplK, rpsA and rpsU harboured stalled RNAP in vivo; this finding suggests that RNAP stalling after initiation is widespread in E. coli. Consistent with the characteristics of both abortive and promoter-proximal σ70-dependent paused complexes, RNAP trapping at most of the newly identified stall sites was eliminated by the rpoDL402Fσ70 mutational alteration and by site mutations, and was enhanced by GreA deficiency. In addition to promoter-proximal RNAP trapping, we observed transcription-dependent DNA modifications spanning the tnaA and cspA leader regions up to 100 bp downstream of the transcription start site.