Bacteriophage T4 MotA Activator and the β-Flap Tip of RNA Polymerase Target the Same Set of σ70 Carboxyl-terminal Residues

Sigma factors, the specificity subunits of RNA polymerase, are involved in interactions with promoter DNA, the core subunits of RNA polymerase, and transcription factors. The bacteriophage T4-encoded activator, MotA, is one such factor, which engages the C terminus of the Escherichia coli housekeeping sigma factor, sigma(70). MotA functions in concert with a phage-encoded co-activator, AsiA, as a molecular switch. This process, termed sigma appropriation, inhibits host transcription while activating transcription from a class of phage promoters. Previous work has demonstrated that MotA contacts the C terminus of sigma(70), H5, a region that is normally bound within RNA polymerase by its interaction with the beta-flap tip. To identify the specific sigma(70) residues responsible for interacting with MotA and the beta-flap tip, we generated single substitutions throughout the C terminus of sigma(70). We find that MotA targets H5 residues that are normally engaged by the beta-flap. In two-hybrid assays, the interaction of sigma(70) with either the beta-flap tip or MotA is impaired by alanine substitutions at residues Leu-607, Arg-608, Phe-610, Leu-611, and Asp-613. Transcription assays identify Phe-610 and Leu-611 as the key residues for MotA/AsiA-dependent transcription. Phe-610 is a crucial residue in the H5/beta-flap tip interaction using promoter clearance assays with RNA polymerase alone. Our results show how the actions of small transcriptional factors on a defined local region of RNA polymerase can fundamentally change the specificity of polymerase.