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

Michiko M. Nakano, Ann Lin, Cole S. Zuber, Kate J. Newberry, Richard G. Brennan, Peter Zuber

Research output: Contribution to journalArticle

39 Scopus citations

Abstract

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.

Original languageEnglish (US)
Article numbere8664
JournalPloS one
Volume5
Issue number1
DOIs
StatePublished - Jan 13 2010

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

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