RESUMO
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.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Elementos de Resposta , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação Transcricional , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Sequência de Bases , Sítios de Ligação , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas , Tiorredoxina Dissulfeto Redutase/genética , Tiorredoxina Dissulfeto Redutase/metabolismo , Fatores de Transcrição/genéticaRESUMO
While dnaK and tig are the essential components for nascent polypeptide folding in E. coli, deletion did not confer synthetic lethality in B. subtilis, suggesting that under normal growth conditions, another system or mechanism with a specific role prevails. Likewise, survival at high temperature suffered dramatically, resulting from deletion of several sets of heat shock genes, thus during sudden stress various heat shock genes act synergistically to protect the proteins.