The interaction between σS, the stationary phase σ factor, and the core enzyme of Escherichia coli RNA polymerase

Background: The RNA polymerase holoenzyme of Escherichia coli is composed of a core enzyme (subunit structure alpha(2)betabeta') associated with one of the sigma subunits, required for promoter recognition. Different sigma factors compete for core binding. Among the seven sigma factors present in E. coli, sigma(70) controls gene transcription during the exponential phase, whereas sigma(s) regulates the transcription of genes in the stationary phase or in response to different stresses. Using labelled sigma(s) and sigma(70), we compared the affinities of both sigma 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 sigma(s) binds to the core enzyme with fivefold reduced affinity compared to sigma(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 sigma(s), while increasing the accessibility in region 4.1 of sigma(s). We have also analysed the positioning of sigma(s) on the holoenzyme by the proximity-dependent protein cleavage method using sigma(s) derivatives in which FeBABE was tethered to single cysteine residues at nine different positions. Protein cutting patterns are observed on the beta and beta' subunits, but not alpha. Regions 2.5, 3.1 and 3.2 of sigma(s) are close to both beta and beta' subunits, in agreement with iron chelate protein footprinting data. Conclusions: A comparison between these results using sigma(s) and previous data from sigma(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.