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

Abstract

ABSTRACT A number of transcriptional regulators mediate their effects through direct contact with the ς 70 subunit of Escherichia coli RNA polymerase (RNAP). In particular, several regulators have been shown to contact a C-terminal portion of ς 70 that harbors conserved region 4. This region of ς contains a putative helix-turn-helix DNA-binding motif that contacts the −35 element of ς 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-ς factor Rsd and the ς 70 subunit of E. coli RNAP. Using this system, we found that Rsd can interact with an 86-amino-acid C-terminal fragment of ς 70 and also that amino acid substitution R596H, within region 4 of ς 70 , weakens this interaction. We demonstrated the specificity of this effect by showing that substitution R596H does not weaken the interaction between ς and two other regulators shown previously to contact region 4 of ς 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 ς 70 subunit of RNAP from this organism. We found that amino acid substitution R600H in ς 70 from P. aeruginosa , corresponding to the R596H substitution in E. coli ς 70 , specifically weakens the interaction between AlgQ and ς 70 . Taken together, our findings suggest that Rsd and AlgQ contact similar surfaces of RNAP present in region 4 of ς 70 and probably regulate gene expression through this contact.