ExsA Recruits RNA Polymerase to an Extended -10 Promoter by Contacting Region 4.2 of Sigma-70

ExsA is a member of the AraC family of transcriptional activators and is required for expression of the Pseudomonas aeruginosa type III secretion system (T3SS). ExsA-dependent promoters consist of two binding sites for monomeric ExsA located approximately 50 bp upstream of the transcription start sites. Binding to both sites is required for recruitment of sigma(70)-RNA polymerase (RNAP) to the promoter. ExsA-dependent promoters also contain putative -35 hexamers that closely match the sigma(70) consensus but are atypically spaced 21 or 22 bp from the -10 hexamer. Because several nucleotides located within the putative -35 region are required for ExsA binding, it is unclear whether the putative -35 region makes an additional contribution to transcription initiation. In the present study we demonstrate that the putative -35 hexamer is dispensable for ExsA-independent transcription from the P(exsC) promoter and that deletion of sigma(70) region 4.2, which contacts the -35 hexamer, has no effect on ExsA-independent transcription from P(exsC). Region 4.2 of sigma(70), however, is required for ExsA-dependent activation of the P(exsC) and P(exsD) promoters. Genetic data suggest that ExsA directly contacts region 4.2 of sigma(70), and several amino acids were found to contribute to the interaction. In vitro transcription assays demonstrate that an extended -10 element located in the P(exsC) promoter is important for overall promoter activity. Our collective data suggest a model in which ExsA compensates for the lack of a -35 hexamer by interacting with region 4.2 of sigma(70) to recruit RNAP to the promoter.