title: Bacteriophage T4 MotA activator and the β-flap tip of RNA polymerase target the same set of σ70 carboxyl-terminal residues.
abstract: 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, σ(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 σ(70), H5, a region that is normally bound within RNA polymerase by its interaction with the β-flap tip. To identify the specific σ(70) residues responsible for interacting with MotA and the β-flap tip, we generated single substitutions throughout the C terminus of σ(70). We find that MotA targets H5 residues that are normally engaged by the β-flap. In two-hybrid assays, the interaction of σ(70) with either the β-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/β-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.
authors.affiliation: Gene Expression and Regulation Section, Laboratory of Cell and Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Richard P
authors.initials: RP
authors.lastname: Bonocora
authors.forename: Phillip K
authors.initials: PK
authors.lastname: Decker
authors.forename: Stephanie
authors.initials: S
authors.lastname: Glass
authors.forename: Leslie
authors.initials: L
authors.lastname: Knipling
authors.forename: Deborah M
authors.initials: DM
authors.lastname: Hinton
mesh_terms: D017122:Bacteriophage T4 / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism* / Q000821:virology; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000502:physiology; D017434:Protein Structure, Tertiary; D012808:Sigma Factor / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000502:physiology; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C067996:MotA protein, Enterobacteria phage T4; D012808:Sigma Factor; D014157:Transcription Factors; D014764:Viral Proteins; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases