The Bacteriophage T4 Inhibitor and Coactivator AsiA Inhibits Escherichia coli RNA Polymerase More Rapidly in the Absence of σ70 Region 1.1: Evidence that Region 1.1 Stabilizes the Interaction between σ70 and Core

摘要

The N-terminal region (region 1.1) of σ70, the primary σ subunit of Escherichia coli RNA polymerase, is a negatively charged domain that affects the DNA binding properties of σ70 regions 2 and 4. Region 1.1 prevents the interaction of free σ70 with DNA and modulates the formation of stable (open) polymerase/promoter complexes at certain promoters. The bacteriophage T4 AsiA protein is an inhibitor of σ70-dependent transcription from promoters that require an interaction between σ70 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 σ70 region 4. We have investigated the effect of region 1.1 on AsiA inhibition and MotA/AsiA activation. We show that σ70 region 1.1 is not required for MotA/AsiA activation at the T4 middle promoter PuvsX. 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 σ70 that lacks region 1.1 is less stable than polymerase with full-length σ70. Our previous work has demonstrated that the AsiA-inhibited polymerase is formed when AsiA binds to region 4 of free σ70 and then the AsiA/σ70 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 σ70 plus core, yielding more free σ70 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 σ70. 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 σ70 arises through an indirect effect.