Mutational analysis of the Bacillus subtilis RNA polymerase at C-terminal domain supports the interference model of Spx-dependent repression

The Spx protein of Bacillus subtilis exerts both positive and negative transcriptional control in response to oxidative stress by interacting with the C-terminal domain of the RNA polymerase (RNAP) alpha subunit (alpha CTD). Thus, transcription of the srf operon at the onset of competence development, which requires the ComA response regulator of the ComPA signal transduction system, is repressed by Spx-alpha CTD interaction. Previous genetic and structural analyses have determined that an Spx-binding surface resides in and around the alpha 1 region of alpha CTD. Alanine-scanning mutagenesis of B. subtilis oiCTD uncovered residue positions required for Spx function and ComA-dependent srf transcriptional activation. Analysis of srf-lacZ fusion expression, DNase I footprinting, and solid-phase promoter retention experiments indicate that Spx interferes with ComA-alpha CTD interaction and that residues Y263, C265, and K267 of the oil region lie within overlapping ComA- and Spx-binding sites for uCTD interaction. Evidence is also presented that oxidized Spx, while enhancing interference of activator-RNAP interaction, is not essential for negative control.