The bacteriophage T4 AsiA protein: a molecular switch for sigma 70-dependent promoters.
The AsiA protein, encoded by bacteriophage T4, inhibits Esigma70-dependent transcription at bacterial and early-phage promoters. We demonstrate that the inhibitory action of AsiA involves interference with the recognition of the -35 consensus promoter sequence by host RNA polymerase. In vitro experiments were performed with a C-terminally labelled sigma factor that is competent for functional holoenzyme reconstitution. By protease and hydroxyl radical protein footprinting, we show that AsiA binds region 4.2 of sigma70, which recognizes the -35 sequence. Direct interference with the recognition of the promoter at this locus is supported by two parallel experiments. The stationary-phase sigma factor containing holoenzyme, which can initiate transcription at promoters devoid of a -35 region, is insensitive to AsiA inhibition. The recognition of a galP1 promoter by Esigma70 is not affected by the presence of AsiA. Therefore, we conclude that AsiA inhibits transcription from Escherichia coli and T4 early promoters by counteracting the recognition of region 4.2 of sigma70 with the -35 hexamer.
Colland
F
F
Unité de Physicochimie des Macromolécules Biologiques, URA 1149 du CNRS, Institut Pasteur, Paris, France.
Orsini
G
G
Brody
E N
EN
Buc
H
H
Kolb
A
A
D000595:Amino Acid Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D018983:DNA Footprinting; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004586:Electrophoresis / Q000379:methods; D019077:Helix-Turn-Helix Motifs; D017665:Hydroxyl Radical / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012808:Sigma Factor / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
C078876:AsiA protein, Enterobacteria phage T4; D011994:Recombinant Proteins; D012808:Sigma Factor; D014764:Viral Proteins; D017665:Hydroxyl Radical; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I