title: Determinants of affinity and activity of the anti-sigma factor AsiA.
abstract: The AsiA protein is a T4 bacteriophage early gene product that regulates transcription of host and viral genes. Monomeric AsiA binds tightly to the sigma(70) subunit of Escherichia coli RNA polymerase, thereby inhibiting transcription from bacterial promoters and phage early promoters and coactivating transcription from phage middle promoters. Results of structural studies have identified amino acids at the protomer-protomer interface in dimeric AsiA and at the monomeric AsiA-sigma(70) interface and demonstrated substantial overlap in the sets of residues that comprise each. Here we evaluate the contributions of individual interfacial amino acid side chains to protomer-protomer affinity in AsiA homodimers, to monomeric AsiA affinity for sigma(70), and to AsiA function in transcription. Sedimentation equilibrium, dynamic light scattering, electrophoretic mobility shift, and transcription activity measurements were used to assess affinity and function of site-specific AsiA mutants. Alanine substitutions for solvent-inaccessible residues positioned centrally in the protomer-protomer interface of the AsiA homodimer, V14, I17, and I40, resulted in the largest changes in free energy of dimer association, whereas alanine substitutions at other interfacial positions had little effect. These residues also contribute significantly to AsiA-dependent regulation of RNA polymerase activity, as do additional residues positioned at the periphery of the interface (K20 and F21). Notably, the relative contributions of a given amino acid side chain to RNA polymerase inhibition and activation (MotA-independent) by AsiA are very similar in most cases. The mainstay for intermolecular affinity and AsiA function appears to be I17. Our results define the core interfacial residues of AsiA, establish roles for many of the interfacial amino acids, are in agreement with the tenets underlying protein-protein interactions and interfaces, and will be beneficial for a general, comprehensive understanding of the mechanistic underpinnings of bacterial RNA polymerase regulation.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA.
authors.forename: Joshua M
authors.initials: JM
authors.lastname: Gilmore
authors.forename: Ramona J
authors.initials: RJ
authors.lastname: Bieber Urbauer
authors.forename: Leonid
authors.initials: L
authors.lastname: Minakhin
authors.forename: Vladimir
authors.initials: V
authors.lastname: Akoyev
authors.forename: Michal
authors.initials: M
authors.lastname: Zolkiewski
authors.forename: Konstantin
authors.initials: K
authors.lastname: Severinov
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Urbauer
mesh_terms: D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors / Q000737:chemistry; D015967:Gene Expression Regulation, Viral; D008027:Light; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D055503:Protein Multimerization; D012542:Scattering, Radiation; D012808:Sigma Factor / Q000037:antagonists & inhibitors / Q000737:chemistry; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: C078876:AsiA protein, Enterobacteria phage T4; D012808:Sigma Factor; D014764:Viral Proteins; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases