Molecular basis for RNA polymerase-dependent transcription complex recycling by the helicase-like motor protein HelD

Timothy P Newing,Catherine J Dawson,Peter J Lewis,Michael Miller,Aaron J Oakley,Simon H J Brown,Gökhan Tolun,James C Bouwer

doi:10.1038/s41467-020-20157-5

Abstract

In bacteria, transcription complexes stalled on DNA represent a major source of roadblocks for the DNA replication machinery that must be removed in order to prevent damaging collisions. Gram-positive bacteria contain a transcription factor HelD that is able to remove and recycle stalled complexes, but it was not known how it performed this function. Here, using single particle cryo-electron microscopy, we have determined the structures of Bacillus subtilis RNA polymerase (RNAP) elongation and HelD complexes, enabling analysis of the conformational changes that occur in RNAP driven by HelD interaction. HelD has a 2-armed structure which penetrates deep into the primary and secondary channels of RNA polymerase. One arm removes nucleic acids from the active site, and the other induces a large conformational change in the primary channel leading to removal and recycling of the stalled polymerase, representing a novel mechanism for recycling transcription complexes in bacteria.

Highlights

In bacteria, transcription complexes stalled on DNA represent a major source of roadblocks for the DNA replication machinery that must be removed in order to prevent damaging collisions
We show this is not the case and that the structure of HelD enables an unusual mode of transcription complex recycling involving a large conformational change in RNA polymerase (RNAP)
Using single particle cryo-electron microscopy, we determined the structures of Bacillus subtilis RNAP elongation and HelD complexes, enabling analysis of the conformational changes that occur in RNAP driven by HelD interaction

Summary

Introduction

Transcription complexes stalled on DNA represent a major source of roadblocks for the DNA replication machinery that must be removed in order to prevent damaging collisions. Using single particle cryo-electron microscopy, we have determined the structures of Bacillus subtilis RNA polymerase (RNAP) elongation and HelD complexes, enabling analysis of the conformational changes that occur in RNAP driven by HelD interaction. One arm removes nucleic acids from the active site, and the other induces a large conformational change in the primary channel leading to removal and recycling of the stalled polymerase, representing a novel mechanism for recycling transcription complexes in bacteria. Transcription is highly sensitive to DNA damage, which causes the elongation complex (EC) to pause, and multiple redundant systems have evolved to ensure rapid removal of RNAP and/or the repair of damaged DNA6–10 This reduces the chance of replication forks colliding with stalled transcription complexes whilst serving as an efficient system for maintaining genome integrity, especially within coding regions. HelD is a prototypical member of a widely dispersed and divergent branch of the SF1 helicase family that maintain genome integrity by removing non-productive transcription roadblocks

Methods

Results

Conclusion