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

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