The Bacteriophage T4 Inhibitor and Coactivator AsiA InhibitsEscherichia coliRNA Polymerase More Rapidly in the Absence of σ70Region 1.1: Evidence that Region 1.1 Stabilizes the Interaction between σ70and Core

Abstract

The N-terminal region (region 1.1) of sigma70, the primary sigma subunit of Escherichia coli RNA polymerase, is a negatively charged domain that affects the DNA binding properties of sigma70 regions 2 and 4. Region 1.1 prevents the interaction of free sigma70 with DNA and modulates the formation of stable (open) polymerase/promoter complexes at certain promoters. The bacteriophage T4 AsiA protein is an inhibitor of sigma70-dependent transcription from promoters that require an interaction between sigma70 region 4 and the -35 DNA element and is the coactivator of transcription at T4 MotA-dependent promoters. Like AsiA, the T4 activator MotA also interacts with sigma70 region 4. We have investigated the effect of region 1.1 on AsiA inhibition and MotA/AsiA activation. We show that sigma70 region 1.1 is not required for MotA/AsiA activation at the T4 middle promoter P(uvsX). However, the rate of AsiA inhibition and of MotA/AsiA activation of polymerase is significantly increased when region 1.1 is missing. We also find that RNA polymerase reconstituted with sigma70 that lacks region 1.1 is less stable than polymerase with full-length sigma70. Our previous work has demonstrated that the AsiA-inhibited polymerase is formed when AsiA binds to region 4 of free sigma70 and then the AsiA/sigma70 complex binds to core. Our results suggest that in the absence of region 1.1, there is a shift in the dynamic equilibrium between polymerase holoenzyme and free sigma70 plus core, yielding more free sigma70 at any given time. Thus, the rate of AsiA inhibition and AsiA/MotA activation increases when RNA polymerase lacks region 1.1 because of the increased availability of free sigma70. Previous work has argued both for and against a direct interaction between regions 1.1 and 4. Using an E. coli two-hybrid assay, we do not detect an interaction between these regions. This result supports the idea that the ability of region 1.1 to prevent DNA binding by free sigma70 arises through an indirect effect.