Nascent RNA sequencing reveals σ70-dependent pausing of elongating RNA polymerase surrounding transcription start sites

However, the prevalence, mechanisms, and regulatory functions of σ70-dependent promoter-distal pausing remain poorly understood. Here, we optimized RNase I footprinting coupled with nascent elongating transcript sequencing (RNET-seq) by stabilizing σ70-retaining transcription elongation complexes, enabling genome-wide identification of σ70-dependent pause sites relative to transcription start sites (TSSs).

RNET-seq revealed a substantial fraction of promoter-distal pause sites located within the −1 to +2-bp region surrounding TSSs, which we termed G0 pausing. The −10 element, but not the −35 element, was critical for G0 pausing, and the strength of the −10 element was positively correlated with σ70-dependent RNAP retention. In addition, the promoter discriminator element acted as a backtracking signal that promoted backtracking of paused transcription elongation complexes at G0 sites.