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

Abstract The Rhodobacter sphaeroides extra cytoplasmic function sigma factor, σ E , directs transcription of promoters for the cycA gene ( cycA P3) and the rpoEchrR operon ( rpoE P1). These genes encode the periplasmic electron carrier cytochrome c 2 and σ E /ChrR, respectively. Using in vitro transcription assays with purified R . sphaeroides core RNA polymerase and σ E , we show that ChrR is sufficient to inhibit σ E -dependent transcription. Inhibition is proposed to proceed through a binding interaction, since σ E and ChrR form a 1:1 complex that can be purified when expressed at high levels in Escherichia coli . Active preparations of ChrR and the σ E /ChrR complex each contain stoichiometric zinc. Removal of zinc from ChrR or a single amino acid substitution that abolishes zinc binding, results in a protein that is incapable of inhibiting σ E activity or forming a complex with the sigma factor, indicating that metal binding is important to ChrR activity. Treatment of ChrR with the thiol-modifying reagent p -hydroxymecuriphenylsulfonic acid results in the release of about one mole of zinc per mole of protein. Furthermore, two N-terminal cysteine residues are protected from reaction with the thiol-specific reagent dithionitrobenzoic acid until zinc is removed, suggesting that these residues may be involved in zinc binding. These data indicate that ChrR is a specific anti-sigma factor of σ E that requires zinc for function. Based on amino acid sequence similarity, we propose that ChrR is part of a family of similar anti-sigma factors that are found in α and γ proteobacteria.