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, σE, directs transcription of promoters for the cycA gene (cycA P3) and the rpoEchrR operon (rpoE P1). These genes encode the periplasmic electron carrier cytochrome c2 and σE/ChrR, respectively. Using in vitro transcription assays with purified R. sphaeroides core RNA polymerase and σE, we show that ChrR is sufficient to inhibit σE-dependent transcription. Inhibition is proposed to proceed through a binding interaction, since σE 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 σE/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 σE 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 σE 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.