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

Sarah K Stumper,Robert Landick,Harini Ravi,Anne Gershenson,Jeff Gelles,Ivan R Corrêa,Rachel Anne Mooney,Larry J Friedman

doi:10.7554/elife.40576

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.

Highlights

Multi-subunit RNA polymerases (RNAPs), the molecular machines responsible for the synthesis of cellular mRNAs, contain catalytic subunits that are conserved across all domains of life (Cramer, 2002; Werner and Grohmann, 2011)
We show that GreB repression of initiation at a ribosomal RNA (rRNA) promoter falls into the latter category
We show by direct observation that GreB binds to free s70RNAP in solution and its presence on RNAP does not affect the formation of initial closed complexes, their stabilities, or their rates of dissociation (Figure 5)

Summary

Introduction

Multi-subunit RNA polymerases (RNAPs), the molecular machines responsible for the synthesis of cellular mRNAs, contain catalytic subunits that are conserved across all domains of life (Cramer, 2002; Werner and Grohmann, 2011). In addition to their conserved catalytic machinery, RNAPs contain conserved binding sites for regulatory proteins. GreB stimulates cleavage of RNA that has reverse threaded through the active site (backtracked) by multiple nucleotides, for instance after nucleotide misincorporation at the RNA 30 end (Borukhov et al, 2001; Borukhov et al, 1993; Laptenko et al, 2003). By causing removal of the backtracked segment of the nascent transcript both GreB and its paralog, GreA, can stimulate transcription by aiding

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Results

Conclusion