Phage T4 early promoters are resistant to inhibition by the anti-sigma factor AsiA.
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.
Orsini
Gilbert
G
Unité des Régulations Transcriptionnelles, Département de Microbiologie Fondamentale et Médicale, URA 2185 du CNRS, Institut Pasteur, F-75724 Paris Cedex 15, France.
Igonet
Sébastien
S
Pène
Carole
C
Sclavi
Bianca
B
Buckle
Malcolm
M
Uzan
Marc
M
Kolb
Annie
A
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*
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