PMID:21911499
| Citation |
Bonocora, RP, Decker, PK, Glass, S, Knipling, L and Hinton, DM (2011) Bacteriophage T4 MotA activator and the β-flap tip of RNA polymerase target the same set of σ70 carboxyl-terminal residues. J. Biol. Chem. 286:39290-6 |
|---|---|
| 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. |
| Links |
PubMed PMC3234753 Online version:10.1074/jbc.M111.278762 |
| Keywords |
Bacteriophage T4/genetics; Bacteriophage T4/metabolism; DNA-Binding Proteins/genetics; DNA-Binding Proteins/metabolism; DNA-Directed RNA Polymerases/genetics; DNA-Directed RNA Polymerases/metabolism; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli/virology; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Promoter Regions, Genetic/physiology; Protein Structure, Tertiary; Sigma Factor/genetics; Sigma Factor/metabolism; Substrate Specificity; Transcription Factors/genetics; Transcription Factors/metabolism; Transcription, Genetic/physiology; Viral Proteins/genetics; Viral Proteins/metabolism |
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