Promoter recognition by a complex of Spx and the C-terminal domain of the RNA polymerase α subunit

Journal Article

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 SpxC10S 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 SpxG52R mutant, defective in αCTD binding, did not interact with the αCTD-trxB complex. Spx R60E not only failed to complex with aCTD-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. © 2010 Nakano et al.

Full Text

Duke Authors

Cited Authors

  • Nakano, MM; Lin, A; Zuber, CS; Newberry, KJ; Brennan, RG; Zuber, P

Published Date

  • 2010

Published In

Volume / Issue

  • 5 / 1

International Standard Serial Number (ISSN)

  • 1932-6203

Digital Object Identifier (DOI)

  • 10.1371/journal.pone.0008664