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

Abstract

ABSTRACT 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 σ 70 -RNA polymerase (RNAP) to the promoter. ExsA-dependent promoters also contain putative −35 hexamers that closely match the σ 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 σ 70 region 4.2, which contacts the −35 hexamer, has no effect on ExsA-independent transcription from P exsC . Region 4.2 of σ 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 σ 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 σ 70 to recruit RNAP to the promoter.