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Mol Microbiol. 2008 Aug;69(3):765-79. doi: 10.1111/j.1365-2958.2008.06330.x.

Activation of transcription initiation by Spx: formation of transcription complex and identification of a Cis-acting element required for transcriptional activation.

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  • 1Department of Environmental and Biomolecular Systems, OGI School of Science and Engineering, Oregon Health and Science University, 20000 NW Walker Rd., Beaverton, OR 97006, USA. pzuber@ebs.ogi.edu

Abstract

The Spx protein of Bacillus subtilis interacts with RNA polymerase (RNAP) to activate transcription initiation in response to thiol-oxidative stress. Protein-DNA cross-linking analysis of reactions containing RNAP, Spx and trxA (thioredoxin) or trxB (thioredoxin reductase) promoter DNA was undertaken to uncover the organization of the Spx-activated transcription initiation complex. Spx induced contact between the RNAP sigma(A) subunit and the -10 promoter sequence of trxA and B, and contact of the betabeta' subunits with core promoter DNA. No Spx-DNA contact was detected. Spx mutants, Spx(C10A) and Spx(G52R.), or RNAP alpha C-terminal domain mutants that impair productive Spx-RNAP interaction did not induce heightened sigma and betabeta' contact with the core promoter. Deletion analysis and the activity of hybrid promoter constructs having upstream trxB DNA fused at positions -31, -36 and -41 of the srf (surfactin synthetase) promoter indicated that a cis-acting site between -50 and -36 was required for Spx activity. Mutations at -43 and -44 of trxB abolished Spx-dependent transcription and Spx-induced cross-linking between the sigma subunit and the -10 region. These data are consistent with a model that Spx activation requires contact between the Spx/RNAP complex and upstream promoter DNA, which allows Spx-induced engagement of the sigma and large subunits with the core promoter.

PMID:
18687074
[PubMed - indexed for MEDLINE]
PMCID:
PMC2758557
Free PMC Article
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