The displacement of the σ70finger in initial transcription is highly heterogeneous and promoter-dependent

ABSTRACT Bacterial sigma factors (σ) contain a highly conserved structural module, the ‘‘σ-finger’’, which forms a loop that protrudes towards the RNA polymerase (RNAP) active-centre in the open complex and has been implicated in pre-organisation 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. We find that the σ-finger is displaced from its position inside the active site cleft before promoter escape, and after synthesis of RNAs with lengths that are highly dependent on the sequence of the promoter used. Real-time smFRET measurements reveal the presence of significant heterogeneity in the timing of finger displacement and show that different σ-finger conformations in single open transcription complexes are associated with substantially different kinetics in transcription initiation and promoter escape, potentially impacting gene regulation in bacteria.