Fig. 3.
Mutations that weaken the σ70 region 4/β-flap interaction restore sensitivity of Eσ70 F563Y to AsiA. Shown are the results of single round in vitro transcription assays performed on T7A2 promoter in the absence or presence of increasing concentrations of AsiA (50 or 100 nM) by using wild-type or mutant RNAP holoenzymes. For each RNAP, the transcripts were quantified (by imagequant) to determine the magnitude of the inhibitory effect of AsiA. Thus, the amount of transcript produced in the absence of AsiA was set at 100% for each holoenzyme. Note that all of the mutant holoenzymes except those containing σ70 with both the R541C and L607P substitutions initiated transcription from T7A2 approximately as well as Eσ70 WT (compare lanes 4, 7, 10, 16, and 19 with lane 1). Eσ70 R541C/L607P and Eσ70 F563Y/R541C/L607P initiated transcription from T7A2 only ≈50% as efficiently as Eσ70 WT. Disruption of the interaction of σ70 with the β-flap is expected to cause defects in the recognition of –10/–35 promoters, and analysis of the effects of the R541C and L607P substitutions indicated that it was necessary to introduce both of these substitutions into σ70 to detect a significant defect in the recognition of strong –10/–35 promoters in vitro (B.E.N., unpublished data). No defects in the recognition of a control extended –10 promoter were detected with σ70 mutants bearing substitutions R541C and L607P either singly or in combination (B.E.N., unpublished data).