Delayed inhibition mechanism for secondary channel factor regulation of ribosomal RNA transcription

Author:

Stumper Sarah K1,Ravi Harini1,Friedman Larry J1ORCID,Mooney Rachel Anne2,Corrêa Ivan R3,Gershenson Anne4ORCID,Landick Robert25ORCID,Gelles Jeff1ORCID

Affiliation:

1. Department of Biochemistry, Brandeis University, Waltham, United States

2. Department of Biochemistry, University of Wisconsin, Madison, United States

3. New England Biolabs, Ipswich, United States

4. Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, United States

5. Department of Bacteriology, University of Wisconsin, Madison, United States

Abstract

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.

Funder

National Institute of General Medical Sciences

National Science Foundation

Publisher

eLife Sciences Publications, Ltd

Subject

General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology,General Medicine,General Neuroscience

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