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.
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MESH: Bacteriophage T4; MESH: Base Sequence; MESH: DNA-Directed RNA Polymerases; MESH: Endodeoxyribonucleases; MESH: Escherichia coli; MESH: Molecular Sequence Data; MESH: Multienzyme Complexes; MESH: Point Mutation; MESH: Promoter Regions (Genetics); MESH: Protein Binding; MESH: Sigma Factor; MESH: Transcription Factors; MESH: Transcription, Genetic; MESH: Viral Proteins; MESH: DNA Footprinting; MESH: DNA-Binding Proteins; [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology
Journal articles; Life Sciences [q-bio]; Ecole Normale Supérieure de Cachan; Institut Jacques Monod; Laboratoire de Biotechnologie et Pharmacologie génétique Appliquée; CNRS - Centre national de la recherche scientifique; Université Denis Diderot - Paris VII; Université Sorbonne Paris Cité; Institut Pasteur
ISSN: 0950-382X; EISSN: 1365-2958; Molecular Microbiology; https://hal.archives-ouvertes.fr/hal-00277452; Molecular Microbiology, Wiley, 2004, 52 (4), pp.1013-28. ⟨10.1111/j.1365-2958.2004.04038.x⟩