Promoter Recognition by a Complex of Spx and the C-Terminal Domain of the RNA Polymerase α Subunit

Background Spx, an ArsC (arsenate reductase) family member, is a global transcriptional regulator of the microbial stress response and is highly conserved amongst Gram-positive bacteria. Bacillus subtilis Spx protein exerts positive and negative control of transcription through its interaction with the C-terminal domain of the RNA polymerase (RNAP) α subunit (αCTD). Spx activates trxA (thioredoxin) and trxB (thioredoxin reductase) in response to thiol stress, and bears an N-terminal C10XXC13 redox disulfide center that is oxidized in active Spx. Methodology/Principal Findings The structure of mutant Spx C10S showed a change in the conformation of helix α4. Amino acid substitutions R60E and K62E within and adjacent to helix α4 conferred defects in Spx-activated transcription but not Spx-dependent repression. Electrophoretic mobility-shift assays showed αCTD interaction with trxB promoter DNA, but addition of Spx generated a supershifted complex that was disrupted in the presence of reductant (DTT). Interaction of αCTD/Spx complex with promoter DNA required the cis -acting elements -45AGCA-42 and -34AGCG-31 of the trxB promoter. The Spx G52R mutant, defective in αCTD binding, did not interact with the αCTD- trxB complex. Spx R60E not only failed to complex with αCTD- trxB , but also disrupted αCTD- trxB DNA interaction. Conclusions/Significance The results show that Spx and αCTD form a complex that recognizes the promoter DNA of an Spx-controlled gene. A conformational change during oxidation of Spx to the disulfide form likely alters the structure of Spx α helix α4, which contains residues that function in transcriptional activation and αCTD/Spx-promoter interaction. The results suggest that one of these residues, R60 of the α4 region of oxidized Spx, functions in αCTD/Spx-promoter contact but not in αCTD interaction.