2026-10-13T00:48:41Z https://www.scidb.cn/oai

10.57760/sciencedb.012c5 2026-09-20T10:10:40Z

2026-09-20 Nascent RNA sequencing reveals σ70-dependent pausing of elongating RNA polymerase surrounding transcription start sites doi:10.57760/sciencedb.012c5 en The primary σ factor σ70 can remain associated with RNA polymerase during transcription elongation and is known to promote widespread promoter-proximal pausing in bacteria. 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. In vitro transcription assays showed that GreB partially relieved G0 pausing, whereas in vivo analysis revealed that deletion of the Gre factors redistributed σ70-dependent pausing, potentially through altered σ70 availability and genome-wide redistribution. Functionally, G0 pausing reduced transcriptional readthrough across the corresponding TSSs and was associated with decreased transcription initiation from these promoters. Together, these findings identify widespread σ70-dependent pausing surrounding TSSs and reveal a previously unrecognized mechanism by which σ70-retaining elongation complexes can regulate transcription within bacterial transcription units. σ70-dependent transcriptional pausing; RNET-seq; Transcription elongation; Transcription start sites; G0 pausing Zhe Sun EMBARGO https://creativecommons.org/licenses/by-nc/4.0/ dataset Science Data Bank