RNA polymerases (RNAPs) contain a conserved 'secondary channel' which binds regulatory factors that modulate transcription initiation. In Escherichia coli, the secondary channel factors (SCFs) GreB and DksA both repress ribosomal RNA (rRNA) transcription, but SCF loading and repression mechanisms are unclear. We observed in vitro fluorescently labeled GreB molecules binding to single RNAPs and initiation of individual transcripts from an rRNA promoter. GreB arrived and departed from promoters only in complex with RNAP. GreB did not alter initial RNAP-promoter binding but instead blocked a step after conformational rearrangement of the initial RNAP-promoter complex. Strikingly, GreB-RNAP complexes never initiated at an rRNA promoter; only RNAP molecules arriving at the promoter without bound GreB produced transcript. The data reveal that a model SCF functions by a 'delayed inhibition' mechanism and suggest that rRNA promoters are inhibited by GreB/DksA because their short-lived RNAP complexes do not allow sufficient time for SCFs to dissociate.
All data analyzed for this study are included in the manuscript or in the source data files (doi:10.5281/zenodo.2530159).
Single-molecule source data files from Delayed inhibition mechanism for secondary channel factor regulation of ribosomal RNA transcriptionZenodo, doi:10.5281/zenodo.2530159.
- Jeff Gelles
- Anne Gershenson
- Robert Landick
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Antoine M van Oijen, University of Wollongong, Australia
© 2019, Stumper et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.