Single-Molecule Fret Analysis of Key Protein Conformational Changes During Promoter Escape by RNA Polymerase

Abstract

Bacterial RNA Polymerases (RNAPs) bind to transcription initiation factors called σ factors to start promoter-specific transcription. Within the σ factor lies a highly conserved structural module, the “σ-finger”, which forms a loop that protrudes towards the RNAP active-center and that interacts with the template strand DNA. The close proximity of the σ-finger to the “heart” of transcription is implicated in the pre-organisation of the template strand DNA, in the synthesis of the first short RNAs, and in the pausing of transcription upon synthesis of a 6-mer RNA. However, the σ-finger also blocks entry of the nascent RNA to the RNAP exit channel and must be displaced during initial transcription as the first step in promoter escape. Despite recent structural studies, σ-finger conformational changes during late transcription initiation and promoter escape are still unknown. Here we report a novel single-molecule FRET ruler that monitors the conformational dynamics of the E.coli σ70-finger from the early stages of transcription initiation to promoter escape. Our results using the σ-finger FRET ruler on transcription complexes formed on a derivative of the lac promoter show that the σ-finger adopts three conformations, which interconvert with rates 0.01-1.5s−1. Intriguingly, we find the σ-finger first displaced in complexes trapped in synthesis of abortive transcripts up to a 10-mer RNA. This observation is likely driven by collision of the σ-finger with the 5’-end of the growing nascent RNA. Based on our results, we propose a new model describing the relation of the conformational changes in the σ-finger to the dynamics of transcription initiation and promoter escape. Archaeal and eukaryotic transcription initiation complexes also contain structural modules that block the RNAP exit channel, so it is therefore likely that this model applies to all kingdoms of life.