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Activation of transcription initiation by Spx: formation of transcription complex and identification of a Cis‐acting element required for transcriptional activation
Author(s) -
Reyes Dindo Y.,
Zuber Peter
Publication year - 2008
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2008.06330.x
Subject(s) - biology , transcription (linguistics) , promoter , sigma factor , thioredoxin reductase , thioredoxin , rna polymerase , protein subunit , microbiology and biotechnology , mutant , dna , transcription preinitiation complex , biochemistry , gene , rna , gene expression , philosophy , linguistics
Summary 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 σ A subunit and the −10 promoter sequence of trxA and B , and contact of the ββ′ subunits with core promoter DNA. No Spx–DNA contact was detected. Spx mutants, Spx C10A and Spx G52R. , or RNAP α C‐terminal domain mutants that impair productive Spx–RNAP interaction did not induce heightened σ and ββ′ 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 σ 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 σ and large subunits with the core promoter.