Bacterial Two-Hybrid Analysis of Interactions between Region 4 of the ς 70 Subunit of RNA Polymerase and the Transcriptional Regulators Rsd from Escherichia coli and AlgQ from Pseudomonas aeruginosa

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Abstract
A number of transcriptional regulators mediate their effects through direct contact with the sigma(70) subunit of Escherichia coli RNA polymerase (RNAP). In particular, several regulators have been shown to contact a C-terminal portion of sigma(70) that harbors conserved region 4. This region of sigma 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 interaction 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 substitution R596H, within region 4 of sigma(70), weakens this interaction. We demonstrated the specificity of this effect by showing that substitution R596H does not weaken the interaction between sigma and two other regulators shown previously to contact region 4 of sigma(70). We also demonstrated that AlgQ, a homolog of Rsd that positively regulates virulence gene expression in Pseudomonas aeruginosa, can contact the C-terminal region of the sigma(70) subunit of RNAP from this organism. We found that amino acid substitution R600H in sigma(70) from P. aeruginosa, corresponding to the R596H substitution in E. coli sigma(70), specifically weakens the interaction between AlgQ and sigma(70). Taken together, our findings suggest that Rsd and AlgQ contact similar surfaces of RNAP present in region 4 of sigma(70) and probably regulate gene expression through this contact.