The importance of zinc-binding to the function of Rhodobacter sphaeroides ChrR as an anti-sigma factor

The Rhodobacter sphaeroides extra cytoplasmic function sigma factor, sigma (E), directs transcription of promoters for the cycA gene (cycA P3) and the rpoEchrR operon (rpoE P1). These genes encode the periplasmic electron car rier cytochrome c(2) and sigma (E)/ChrR, respectively. Using in vitro trans cription assays with purified R. sphaeroides core RNA polymerase and sigma (E), we show that ChrR is sufficient to inhibit sigma (E)-dependent transcr iption. Inhibition is proposed to proceed through a binding interaction, si nce sigma (E) and ChrR form a 1:1 complex that can be purified when express ed at high levels in Escherichia coli. Active preparations of ChrR and the sigma (E)/ChrR complex each contain stoichiometric zinc. Removal of zinc fr om ChrR or a single amino acid substitution that abolishes zinc binding, re sults in a protein that is incapable of inhibiting sigma (E) activity or fo rming a complex with the sigma factor, indicating that metal binding is imp ortant to ChrR activity. Treatment of ChrR with the thiol-modifying reagent p-hydroxy-mecuriphenylsulfonic acid results in the release of about one mo le of zinc per mole of protein. Furthermore, two N-terminal cysteine residu es are protected from reaction with the thiol-specific reagent dithionitrob enzoic acid until zinc is removed, suggesting that these residues may be in volved in zinc binding. These data indicate that ChrR is a specific anti-si gma factor of sigma (E) that requires zinc for function. Based on amino aci d sequence similarity, we propose that ChrR is part of a family of similar anti-sigma factors that are found in alpha and gamma proteobacteria. (C) 20 01 Academic Press.