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The interaction between σ S , the stationary phase σ factor, and the core enzyme of Escherichia coli RNA polymerase
Author(s) -
Colland Frédéric,
Fujita Nobuyuki,
Ishihama Akira,
Kolb Annie
Publication year - 2002
Publication title -
genes to cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.912
H-Index - 115
eISSN - 1365-2443
pISSN - 1356-9597
DOI - 10.1046/j.1365-2443.2002.00517.x
Subject(s) - footprinting , biology , protein subunit , rna polymerase , dna footprinting , transcription (linguistics) , specificity factor , escherichia coli , enzyme , biochemistry , gel electrophoresis , microbiology and biotechnology , polymerase , transcription factor , gene , dna binding protein , linguistics , philosophy
Background: The RNA polymerase holoenzyme of Escherichia coli is composed of a core enzyme (subunit structure α 2 ββ′) associated with one of the σ subunits, required for promoter recognition. Different σ factors compete for core binding. Among the seven σ factors present in E. coli , σ 70 controls gene transcription during the exponential phase, whereas σ S regulates the transcription of genes in the stationary phase or in response to different stresses. Using labelled σ S and σ 70 , we compared the affinities of both σ factors for core binding and investigated the structural changes in the different subunits involved in the formation of the holoenzymes. Results: Using native polyacrylamide gel electrophoresis, we demonstrate that σ S binds to the core enzyme with fivefold reduced affinity compared to σ 70 . Using iron chelate protein footprinting, we show that the core enzyme significantly reduces polypeptide backbone solvent accessibility in regions 1.1, 2.5, 3.1 and 3.2 of σ S , while increasing the accessibility in region 4.1 of σ S . We have also analysed the positioning of σ S on the holoenzyme by the proximity‐dependent protein cleavage method using σ S derivatives in which FeBABE was tethered to single cysteine residues at nine different positions. Protein cutting patterns are observed on the β and β′ subunits, but not α. Regions 2.5, 3.1 and 3.2 of σ S are close to both β and β′ subunits, in agreement with iron chelate protein footprinting data. Conclusions: A comparison between these results using σ S and previous data from σ 70 indicates similar contact patterns on the core subunits and similar characteristic changes associated with holoenzyme formation, despite striking differences in the accessibility of regions 4.1 and 4.2.