The Bacteriophage T4 Inhibitor and Coactivator AsiA Inhibits Escherichia coli RNA Polymerase More Rapidly in the Absence of σ 70 Region 1.1: Evidence that Region 1.1 Stabilizes the Interaction between σ 70 and Core

Abstract

ABSTRACT The N-terminal region (region 1.1) of σ 70 , the primary σ subunit of Escherichia coli RNA polymerase, is a negatively charged domain that affects the DNA binding properties of σ 70 regions 2 and 4. Region 1.1 prevents the interaction of free σ 70 with DNA and modulates the formation of stable (open) polymerase/promoter complexes at certain promoters. The bacteriophage T4 AsiA protein is an inhibitor of σ 70 -dependent transcription from promoters that require an interaction between σ 70 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 σ 70 region 4. We have investigated the effect of region 1.1 on AsiA inhibition and MotA/AsiA activation. We show that σ 70 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 σ 70 that lacks region 1.1 is less stable than polymerase with full-length σ 70 . Our previous work has demonstrated that the AsiA-inhibited polymerase is formed when AsiA binds to region 4 of free σ 70 and then the AsiA/σ 70 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 σ 70 plus core, yielding more free σ 70 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 σ 70 . 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 σ 70 arises through an indirect effect.