Bacterial two-hybrid analysis of interactions between region 4 of the sigma(70) subunit of RNA polymerase and the transcriptional regulators Rsd fromEscherichia coli and AlgQ from Pseudomonas aeruginosa

A number of transcriptional regulators mediate their effects through direct contact with the sigma (70) subunit of Escherichia coli RNA polymerase (RN AP). In particular, several regulators have been shown to contact a C-termi nal portion of sigma (70) that harbors conserved region 4. This region of o r contains a putative helix-turn-helix DNA-binding motif that contacts the -35 element of sigma (70)-dependent promoters directly. Here we report the use of a recently developed bacterial two-hybrid system to study the intera ction between the putative anti-sigma factor Rsd and the sigma (70) subunit of E. coli RNAP. Using this system, we found that Rsd can interact with an 86-amino-acid C-terminal fragment of sigma (70) and also that amino acid s ubstitution R596H, within region 4 of sigma (70), weakens this interaction. We demonstrated the specificity of this effect by showing that substitutio n R596H does not weaken the interaction between a and two other regulators shown previously to contact region 4 of sigma (70) We also demonstrated tha t AlgQ, a homolog of Rsd that positively regulates virulence gene expressio n in Pseudomonas aeruginosa, can contact the C-terminal region of the sigma (70) subunit of RNAP from this organism. We found that amino acid substitu tion R600H in sigma (70) from P. aeruginosa, corresponding to the R596H sub stitution in E. Coli sigma (70), specifically weakens the interaction betwe en AlgQ and sigma (70). Taken together, our findings suggest that Rsd and A lgQ contact similar surfaces of RNAP present in region 4 of sigma (70) and probably regulate gene expression through this contact.