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) alpha subunit (alpha CTD). 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 alpha 4. Amino acid substitutions R60E and K62E within and adjacent to helix alpha 4 conferred defects in Spx-activated transcription but not Spx-dependent repression. Electrophoretic mobility-shift assays showed alpha CTD interaction with trxB promoter DNA, but addition of Spx generated a supershifted complex that was disrupted in the presence of reductant (DTT). Interaction of alpha CTD/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 alpha CTD binding, did not interact with the alpha CTD-trxB complex. Spx R60E not only failed to complex with alpha CTD-trxB, but also disrupted alpha CTD-trxB DNA interaction. Conclusions/Significance: The results show that Spx and alpha CTD 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 alpha helix alpha 4, which contains residues that function in transcriptional activation and alpha CTD/Spx-promoter interaction. The results suggest that one of these residues, R60 of the alpha 4 region of oxidized Spx, functions in alpha CTD/Spx-promoter contact but not in alpha CTD interaction.