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

Publication ,  Journal Article
Nakano, MM; Lin, A; Zuber, CS; Newberry, KJ; Brennan, RG; Zuber, P
Published in: PLoS One
January 13, 2010

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. METHODOLOGY/PRINCIPAL FINDINGS: 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/SIGNIFICANCE: 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.

Duke Scholars

Published In

PLoS One

DOI

EISSN

1932-6203

Publication Date

January 13, 2010

Volume

5

Issue

1

Start / End Page

e8664

Location

United States

Related Subject Headings

  • Promoter Regions, Genetic
  • Mutation
  • General Science & Technology
  • DNA-Directed RNA Polymerases
  • Crystallization
  • Bacterial Proteins
  • Bacillus subtilis
 

Citation

APA
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ICMJE
MLA
NLM
Nakano, M. M., Lin, A., Zuber, C. S., Newberry, K. J., Brennan, R. G., & Zuber, P. (2010). Promoter recognition by a complex of Spx and the C-terminal domain of the RNA polymerase alpha subunit. PLoS One, 5(1), e8664. https://doi.org/10.1371/journal.pone.0008664
Nakano, Michiko M., Ann Lin, Cole S. Zuber, Kate J. Newberry, Richard G. Brennan, and Peter Zuber. “Promoter recognition by a complex of Spx and the C-terminal domain of the RNA polymerase alpha subunit.PLoS One 5, no. 1 (January 13, 2010): e8664. https://doi.org/10.1371/journal.pone.0008664.
Nakano MM, Lin A, Zuber CS, Newberry KJ, Brennan RG, Zuber P. Promoter recognition by a complex of Spx and the C-terminal domain of the RNA polymerase alpha subunit. PLoS One. 2010 Jan 13;5(1):e8664.
Nakano, Michiko M., et al. “Promoter recognition by a complex of Spx and the C-terminal domain of the RNA polymerase alpha subunit.PLoS One, vol. 5, no. 1, Jan. 2010, p. e8664. Pubmed, doi:10.1371/journal.pone.0008664.
Nakano MM, Lin A, Zuber CS, Newberry KJ, Brennan RG, Zuber P. Promoter recognition by a complex of Spx and the C-terminal domain of the RNA polymerase alpha subunit. PLoS One. 2010 Jan 13;5(1):e8664.

Published In

PLoS One

DOI

EISSN

1932-6203

Publication Date

January 13, 2010

Volume

5

Issue

1

Start / End Page

e8664

Location

United States

Related Subject Headings

  • Promoter Regions, Genetic
  • Mutation
  • General Science & Technology
  • DNA-Directed RNA Polymerases
  • Crystallization
  • Bacterial Proteins
  • Bacillus subtilis