title: Phage T4 early promoters are resistant to inhibition by the anti-sigma factor AsiA.
abstract: Phage T4 early promoters are transcribed in vivo and in vitro by the Escherichia coli RNA polymerase holoenzyme Esigma(70). We studied in vitro the effects of the T4 anti-sigma(70) factor AsiA on the activity of several T4 early promoters. In single-round transcription, promoters motB, denV, mrh.2, motA wild type and UP element-deleted motA are strongly resistant to inhibition by AsiA. The alpha-C-terminal domain of Esigma(70) is crucial to this resistance. DNase I footprinting of Esigma(70) and Esigma(70)AsiA on motA and mrh.2 shows extended contacts between the holoenzyme with or without AsiA and upstream regions of these promoters. A TG --> TC mutation of the extended -10 motif in the motA UP element-deleted promoter strongly increases susceptibility to inhibition by AsiA, but has no effect on the motA wild-type promoter: either the UP element or the extended -10 site confers resistance to AsiA. Potassium permanganate reactivity shows that the two structure elements are not equivalent: with AsiA, the motA UP element-deleted promoter opens more slowly whereas the motA TC promoter opens like the wild type. Changes in UV laser photoreactivity at position +4 on variants of motA reveal an analogous distinction in the roles of the extended -10 and UP promoter elements.
authors.affiliation: Unité des Régulations Transcriptionnelles, Département de Microbiologie Fondamentale et Médicale, URA 2185 du CNRS, Institut Pasteur, F-75724 Paris Cedex 15, France.
authors.forename: Gilbert
authors.initials: G
authors.lastname: Orsini
authors.forename: Sébastien
authors.initials: S
authors.lastname: Igonet
authors.forename: Carole
authors.initials: C
authors.lastname: Pène
authors.forename: Bianca
authors.initials: B
authors.lastname: Sclavi
authors.forename: Malcolm
authors.initials: M
authors.lastname: Buckle
authors.forename: Marc
authors.initials: M
authors.lastname: Uzan
authors.forename: Annie
authors.initials: A
authors.lastname: Kolb
mesh_terms: D017122:Bacteriophage T4 / Q000235:genetics / Q000502:physiology*; D001483:Base Sequence; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000502:physiology; D004706:Endodeoxyribonucleases / Q000235:genetics; D004926:Escherichia coli / Q000502:physiology / Q000821:virology; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012808:Sigma Factor / Q000502:physiology; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000502:physiology*
chemical_list: C078876:AsiA protein, Enterobacteria phage T4; D004268:DNA-Binding Proteins; C117370:MRH protein, Enterobacteria phage T4; C067996:MotA protein, Enterobacteria phage T4; D009097:Multienzyme Complexes; D012808:Sigma Factor; D014157:Transcription Factors; D014764:Viral Proteins; C032219:UV endonuclease; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; D004706:Endodeoxyribonucleases