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 sigma(70)-containing Escherichia coli RNA polymerase, the T4 activator MotA, and the T4 co-activator AsiA. T4 middle promoters contain the sigma(70)-10 DNA element. However, these promoters lack the sigma(70)-35 element, having instead a MotA box centered at -30, which is bound by MotA. Previous work has indicated that AsLk and MotA interact with region 4 of sigma(70), the C-terminal portion that normally contacts -35 DNA and the beta-flap structure in core. AsiA binding prevents the sigma(70)/beta-flap and sigma(70)/-35 DNA interactions, inhibiting transcription from promoters that require a -35 element. To test the importance of residues within a region 4 for MotA and AsiA function, we investigated how sigma(70) region 4 mutants interact with AsiA, MotA, and the beta-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 beta-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/beta-flap interaction, significantly impair the AsiA/sigma(70) interaction, and eliminate AsiA inhibition. However, the 4.1 mutant sigma(70) is Still fully competent for activation if both MotA and AsiA are present. A previous NMR structure shows AsiA binding to sigma(70) region 4, dramatically distorting regions 4.1 and 4.2 and indirectly changing the conformation of the MotA interaction site at the sigma(70) C terminus. Our analyses provide biochemical relevance for the a 70 residues identified in the structure, indicate that the interaction of AsiA with sigma(70) 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 sigma(70) .Published by Elsevier Ltd.