The bacteriophage T4 inhibitor and coactivator AsiA inhibits Escherichia coli RNA Polymerase more rapidly in the absence of sigma70 region 1.1: evidence that region 1.1 stabilizes the interaction between sigma70 and 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.

FIG. 1.

MotA/AsiA activation of P_uvsX does not require region 1.1 of σ⁷⁰. Polymerase, reconstituted with 0.4 pmol core and 1 pmol of the indicated σ, was preincubated with or without 9 pmol AsiA in 2.15 μl of incubation buffer I for 30°C for 15 min before the addition of 2.35 μl of DNA buffer II containing 0.04 pmol of P_uvsX DNA and, as indicated, 1.9 pmol MotA. After an incubation at 37°C for 10 min, transcription was initiated by the addition of 0.5 μl of NTP solution I. The positions of the P_uvsX and P_minor RNAs on the denaturing polyacrylamide gel are indicated. Quantitation indicated that the relative level of P_uvsX RNA [(level of P_uvsX RNA with MotA/AsiA)/(level of P_uvsX RNA by polymerase alone)] was similar for each σ: 1.6 (Eσ^fl), 1.7 (Eσ^Δ50), 1.9 (Eσ^Δ75), and 2.0 (Eσ^Δ1.1).

FIG. 2.

Preincubation of AsiA with Eσ^Δ1.1 at 4°C inhibits subsequent transcription. AsiA (9 pmol), core (0.4 pmol), and either 1 pmol σ^fl (A) or 1 pmol σ^Δ1.1 (B) were preincubated as indicated, resulting in a final volume of 2.15 μl of incubation buffer I. The protein mixtures were added to 0.02 pmol of P_uvsX DNA in 2.35 μl of DNA buffer I and incubated at 37°C for 20 s before the addition of 0.5 μl of rifampin (300 μg/ml). The positions of the P_uvsX and P_minor RNAs on the denaturing polyacrylamide gel are indicated.

FIG. 3.

AsiA inhibition of Eσ^Δ1.1 is more rapid than its inhibition of Eσ^fl at either 4°C or 30°C. Transcriptions were performed as for Fig. 2A and B, lanes 7 and 8, except that reactions contained 0.5 pmol of the indicated σ and 0.2 pmol of core and AsiA was incubated with Eσ^fl or Eσ^Δ1.1 at 4°C or 30^οC for the indicated time before the start of transcription. The relative level of P_uvsX RNA [(amount of RNA observed after AsiA-plus-Eσ incubation for time t)/(amount of RNA observed without AsiA)] is shown versus the length of time (in min) of the AsiA-plus-Eσ incubation.

FIG. 4.

AsiA inhibits and MotA/AsiA activates polymerase more rapidly in the absence of region 1.1. Reaction mixtures containing the indicated Eσ (reconstituted with 0.05 pmol core plus 0.2 pmol σ) were incubated at 30°C with 3 pmol AsiA in incubation buffer III for the indicated times. Transcription was initiated by mixing aliquots (3.1 μl) with 1.9 μl DNA buffer IV containing 1.9 pmol MotA, 0.01 pmol pDKT90 (P_uvsX plasmid) that had been restricted with BsaAI, and 0.01 pmol pP_wt that had been restricted with SspI. Reactions were incubated at 37°C for 20 s before the addition of 0.5 μl of rifampin at 300 μg/ml. The relative level of P_uvsX RNA is as follows: (amount of P_uvsX RNA observed after AsiA-plus-Eσ incubation for time t − amount of P_uvsX RNA observed without AsiA)/(amount of P_uvsX RNA observed after AsiA-plus-Eσ incubation for 10 min − amount of P_uvsX RNA observed without AsiA). The relative level of P_wt RNA is as follows: (amount of P_wt RNA observed after AsiA-plus-Eσ incubation for time t)/(amount of P_wt RNA observed without AsiA). These values are shown versus the length of time (in min) of the AsiA-plus-Eσ incubation.

FIG. 5.

Addition of free σ^fl converts Eσ^Δ1.1 to Eσ^fl. Eσ^fl or Eσ^Δ1.1, reconstituted by incubating 0.05 pmol of core with 2.0 pmol of the appropriate σ for 15 min at 30°C, was then incubated either with 12 pmol of σ^Δ1.1 at 30°C (top left) or at 4°C (bottom left) or with 12 pmol of σ^fl at 30°C (top right) or at 4°C (bottom right) in 3.1 μl of incubation buffer (bfr) II for the indicated times. (The first time point was taken after an incubation of 15 s.) The protein mixtures were then added to 0.01 pmol of pDKT90 DNA in 1.9 μl of DNA buffer III and incubated at 37°C for 20 s before the addition of 0.5 μl of rifampin at 300 μg/ml.