Mutational Analysis of σ70 Region 4 Needed for Appropriation by the Bacteriophage T4 Transcription Factors AsiA and MotA

Transcriptional activation of bacteriophage T4 middle promoters requires σ70-containing Escherichia coli RNA polymerase, the T4 activator MotA, and the T4 co-activator AsiA. T4 middle promoters contain the σ70 –10 DNA element. However, these promoters lack the σ70 –35 element, having instead a MotA box centered at –30, which is bound by MotA. Previous work has indicated that AsiA and MotA interact with region 4 of σ70, the C-terminal portion that normally contacts –35 DNA and the β-flap structure in core. AsiA binding prevents the σ70/β-flap and σ70/–35 DNA interactions, inhibiting transcription from promoters that require a –35 element. To test the importance of residues within σ70 region 4 for MotA and AsiA function, we investigated how σ70 region 4 mutants interact with AsiA, MotA, and the β-flap and function in transcription assays in vitro. We find that alanine substitutions at residues 584–588 (region 4.2) do not impair the interaction of region 4 with the β-flap or MotA, but they eliminate the interaction with AsiA and prevent AsiA inhibition and MotA/AsiA activation. In contrast, alanine substitutions at 551-552, 554-555 (region 4.1) eliminate the region 4/β-flap interaction, significantly impair the AsiA/σ70 interaction, and eliminate AsiA inhibition. However, the 4.1 mutant σ70 is still fully competent for activation if both MotA and AsiA are present. A previous NMR structure shows AsiA binding to σ70 region 4, dramatically distorting regions 4.1 and 4.2 and indirectly changing the conformation of the MotA interaction site at the σ70 C terminus. Our analyses provide biochemical relevance for the σ70 residues identified in the structure, indicate that the interaction of AsiA with σ70 region 4.2 is crucial for activation, and support the idea that AsiA binding facilitates an interaction between MotA and the far C terminus of σ70.