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Phage T4 early promoters are resistant to inhibition by the anti‐sigma factor AsiA
Author(s) -
Orsini Gilbert,
Igonet Sébastien,
Pène Carole,
Sclavi Bianca,
Buckle Malcolm,
Uzan Marc,
Kolb Annie
Publication year - 2004
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2004.04038.x
Subject(s) - promoter , biology , rna polymerase , sigma factor , microbiology and biotechnology , transcription (linguistics) , escherichia coli , dna footprinting , genetics , gene , gene expression , linguistics , philosophy
Summary Phage T4 early promoters are transcribed in vivo and in vitro by the Escherichia coli RNA polymerase holoenzyme Eσ 70 . We studied in vitro the effects of the T4 anti‐σ 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 α‐C‐terminal domain of Eσ 70 is crucial to this resistance. DNase I footprinting of Eσ 70 and Eσ 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.