Promoter recognition by a complex of Spx and the C-terminal domain of the RNA polymerase alpha 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 (alphaCTD). 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.


RESULTS
The structure of mutant Spx(C10S) showed a change in the conformation of helix alpha4. Amino acid substitutions R60E and K62E within and adjacent to helix alpha4 conferred defects in Spx-activated transcription but not Spx-dependent repression. Electrophoretic mobility-shift assays showed alphaCTD interaction with trxB promoter DNA, but addition of Spx generated a supershifted complex that was disrupted in the presence of reductant (DTT). Interaction of alphaCTD/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 alphaCTD binding, did not interact with the alphaCTD-trxB complex. Spx(R60E) not only failed to complex with alphaCTD-trxB, but also disrupted alphaCTD-trxB DNA interaction.


CONCLUSIONS
The results show that Spx and alphaCTD 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 alpha4, which contains residues that function in transcriptional activation and alphaCTD/Spx-promoter interaction. The results suggest that one of these residues, R60 of the alpha4 region of oxidized Spx, functions in alphaCTD/Spx-promoter contact but not in alphaCTD interaction.
Nakano
Michiko M
MM
Department of Science & Engineering, School of Medicine, Oregon Health & Science University, Beaverton, Oregon, USA.
Lin
Ann
A
Zuber
Cole S
CS
Newberry
Kate J
KJ
Brennan
Richard G
RG
Zuber
Peter
P
D001412:Bacillus subtilis / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D003460:Crystallization; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011401:Promoter Regions, Genetic
D001426:Bacterial Proteins; D012321:DNA-Directed RNA Polymerases; C113275:RNA polymerase alpha subunit