biostudies-literatureWang AUGCUKDBT/Wellcome Trust India AllianceWellcome TrustBiotechnology and Biological Sciences Research Councilgkaf857https://www.ebi.ac.uk/biostudies/studies/S-EPMC12445709biostudies-literatureUnknown53(17)Most bacterial sigma factors (σ) contain a highly conserved structural module, the 'σ-finger', which forms a loop that protrudes towards the RNA polymerase active centre in the open complex and has been implicated in pre-organization of template DNA, abortive initiation of short RNAs, initiation pausing, and promoter escape. Here, we introduce a novel single-molecule FRET (smFRET) assay to monitor σ-finger motions during transcription initiation and promoter escape. By performing real-time smFRET measurements, we determine that for all promoters studied, displacement occurs before promoter escape and can occur either before or after a clash with the extending RNA. We show that the kinetics of σ-finger displacement are highly dependent on the promoter, with implications for transcription kinetics and regulation. Analogous mechanisms may operate in the similar modules present across all kingdoms of life.Nucleic acids researchThe displacement of the σ70 finger in initial transcription is highly heterogeneous and promoter-dependent.PMC12445709BB/M01116X/1IA/I/21/2/505898221610073587110164/Z/15/ZBB/R008655/1212193/Z/18/ZKapanidis ANWang AGrainger DCMukherjee PMazumder AFletcher AfalseThe displacement of the σ70 finger in initial transcription is highly heterogeneous and promoter-dependent.Most bacterial sigma factors (σ) contain a highly conserved structural module, the 'σ-finger', which forms a loop that protrudes towards the RNA polymerase active centre in the open complex and has been implicated in pre-organization of template DNA, abortive initiation of short RNAs, initiation pausing, and promoter escape. Here, we introduce a novel single-molecule FRET (smFRET) assay to monitor σ-finger motions during transcription initiation and promoter escape. By performing real-time smFRET measurements, we determine that for all promoters studied, displacement occurs before promoter escape and can occur either before or after a clash with the extending RNA. We show that the kinetics of σ-finger displacement are highly dependent on the promoter, with implications for transcription kinetics and regulation. Analogous mechanisms may operate in the similar modules present across all kingdoms of life.2025-01-01T00:00:00Z2025 Sep2026-06-04T07:29:44.605Z2026-06-04T03:05:57.715ZS-EPMC124457094096649910.1093/nar/gkaf857