Novel protein--protein interaction between Escherichia coli SoxS and the DNA binding determinant of the RNA polymerase alpha subunit: SoxS functions as a co-sigma factor and redeploys RNA polymerase from UP-element-containing promoters to SoxS-dependent promoters during oxidative stress.
SoxS is the transcription activator of the SoxRS regulon. Despite being synthesized de novo in response to oxidative stress and despite the large disparity between the number of SoxS binding sites and the number of SoxS molecules per cell, SoxS-dependent promoters are rapidly activated after the onset of the stress. With the usual recruitment/post-recruitment mechanisms being unsuitable for activating gene expression under these conditions, we previously proposed that SoxS functions by "pre-recruitment". In pre-recruitment, SoxS forms SoxS-RNA polymerase binary complexes, which use the DNA binding properties of SoxS and sigma(70) to distinguish SoxS-dependent promoters from housekeeping promoters and from the large number of sequence-equivalent but functionally irrelevant SoxS binding sites. With previous work in Escherichia coli having indicated that the most likely target on RNA polymerase for interaction with SoxS is the C-terminal domain of alpha, we investigated the interaction directly with the yeast two-hybrid system. We found that SoxS interacts with the alphaCTD and that SoxS positive control mutations disrupt the interaction. Moreover, single alanine substitutions of the alphaCTD that reduce or enhance SoxS activation in E.coli reduce or enhance the interaction between SoxS and the alphaCTD in yeast. Significantly, the critical amino acid residues lie in and around the DNA binding determinant of the alphaCTD, the first example of an activator contacting this determinant. These interactions were confirmed with an affinity immobilization assay. Lastly, we found that SoxS induction interferes with utilization of the UP element of an rRNA promoter. Thus, by functioning as a co-sigma factor that interacts with the DNA binding determinant of the alphaCTD, SoxS diverts RNA polymerase from UP-containing promoters to SoxS-activatable promoters.
Shah
Ishita M
IM
Department of Biological Sciences, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, USA.
Wolf
Richard E
RE
D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D008957:Models, Genetic; D008958:Models, Molecular; D009154:Mutation; D018384:Oxidative Stress; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012808:Sigma Factor / Q000235:genetics / Q000378:metabolism*; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques
D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D012808:Sigma Factor; D015534:Trans-Activators; C070725:SoxS protein, E coli; D012321:DNA-Directed RNA Polymerases; C113275:RNA polymerase alpha subunit