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

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 σA 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, SpxC¹⁰A and SpxG⁵²R., 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.