title: Interaction of T4 AsiA with its target sites in the RNA polymerase sigma70 subunit leads to distinct and opposite effects on transcription.
abstract: Bacteriophage T4 AsiA is a homodimeric protein that orchestrates a switch from the host and early viral transcription to middle viral transcription by binding to the sigma(70) subunit of Escherichia coli RNA polymerase holoenzyme (Esigma(70)) and preventing promoter complex formation on most E.coli and early T4 promoters. In addition, Esigma(70)AsiA, but not Esigma(70), is a substrate of transcription activation by T4-encoded DNA-binding protein MotA, a co-activator of transcription from middle viral promoters. The molecular determinants of sigma(70)-AsiA interaction necessary for transcription inhibition reside in the sigma(70) conserved region 4.2, which recognizes the -35 promoter consensus element. The molecular determinants of sigma(70)-AsiA interaction necessary for MotA-dependent transcription activation have not been identified. Here, we show that in the absence of sigma(70) region 4.2, AsiA interacts with sigma(70) conserved region 4.1 and activates transcription in a MotA-independent manner. Further, we show that the AsiA dimer must dissociate to interact with either region 4.2 or region 4.1 of sigma(70). We propose that MotA may co-activate transcription by restricting AsiA binding to sigma(70) region 4.1.
authors.affiliation: Department of Genetics, Waksman Institute of Microbiology, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA.
authors.forename: Leonid
authors.initials: L
authors.lastname: Minakhin
authors.forename: Anita
authors.initials: A
authors.lastname: Niedziela-Majka
authors.forename: Konstantin
authors.initials: K
authors.lastname: Kuznedelov
authors.forename: Karen
authors.initials: K
authors.lastname: Adelman
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Urbauer
authors.forename: Tomasz
authors.initials: T
authors.lastname: Heyduk
authors.forename: Konstantin
authors.initials: K
authors.lastname: Severinov
mesh_terms: D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000378:metabolism*; D001665:Binding Sites; D018983:DNA Footprinting; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D005456:Fluorescent Dyes; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D012808:Sigma Factor / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: C078876:AsiA protein, Enterobacteria phage T4; D005456:Fluorescent Dyes; D012808:Sigma Factor; D014764:Viral Proteins; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases