Involvement of region 4 of the σ 70 subunit of RNA polymerase in transcriptional activation of the lux operon during quorum sensing

Abstract

Quorum sensing-dependent activation of the luminescence ( lux) genes of Vibrio fischeri relies on the formation of a complex between the autoinducer molecule, N-(3-oxohexanoyl)- L-homoserine lactone, and the autoinducer-dependent transcriptional activator LuxR. In its active conformation, LuxR binds to a site known as the lux box centered at position −42.5 relative to the luxI transcriptional start site and is thought to function as an ambidextrous activator capable of making multiple contacts with RNA polymerase (RNAP). The specific role of region 4 of the Escherichia coli σ 70 subunit of RNAP in LuxR-dependent activation of the luxI promoter has been investigated. Single-round transcription assays were performed in the presence of purified LuxRΔN, the autoinducer-independent C-terminal domain of LuxR, and a variant RNAP which contained a C-terminally truncated σ 70 subunit devoid of region 4. Results indicated that region 4 is essential for LuxRΔN-dependent luxI transcription, therefore 16 single and two triple alanine substitutions in region 4.2 of σ 70 between amino acid residues 590 and 613 were examined for their effects on LuxR- and LuxRΔN-dependent transcription at the luxI promoter. Taken together, the analyses performed on these variants of RpoD suggest that some individual residues in region 4.2 are important to the mechanism of activator-dependent transcription initiation under investigation.