RESUMO
The bacterial RNA polymerase (RNAP) is a multi-subunit, structurally flexible, complex molecular machine, in which activities associated with DNA opening for transcription-competent open promoter complex (OC) formation reside in the catalytic beta and beta' subunits and the dissociable sigma subunit. OC formation is a multi-step process that involves several structurally conserved mobile modules of beta, beta', and sigma. Here, we present evidence that two flexible modules of beta', the beta' jaw and the beta' clamp and a conserved regulatory Region I domain of sigma(54), jointly contribute to the maintenance of stable DNA strand separation around the trancription start site in OCs formed at sigma(54)-dependent promoters. Clearly, regulated interplay between the mobile modules of the beta' and the sigma subunits of the RNAP appears to be necessary for stable OC formation.
Assuntos
RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , DNA/química , DNA/metabolismo , Escherichia coli/enzimologia , Regiões Promotoras Genéticas/genética , RNA Polimerase Sigma 54/metabolismo , Heparina/farmacologia , Modelos Moleculares , Mutação/genética , Conformação de Ácido Nucleico , Estrutura Terciária de Proteína , Proteínas Repressoras/metabolismo , Sítio de Iniciação de Transcrição , Transcrição GênicaRESUMO
ATP-driven remodelling of initial RNA polymerase (RNAP) promoter complexes occurs as a major post recruitment strategy used to control gene expression. Using a model-enhancer-dependent bacterial system (sigma54-RNAP, Esigma54) and a slowly hydrolysed ATP analogue (ATPgammaS), we provide evidence for a nucleotide-dependent temporal pathway leading to DNA melting involving a small set of sigma54-DNA conformational states. We demonstrate that the ATP hydrolysis-dependent remodelling of Esigma54 occurs in at least two distinct temporal steps. The first detected remodelling phase results in changes in the interactions between the promoter specificity sigma54 factor and the promoter DNA. The second detected remodelling phase causes changes in the relationship between the promoter DNA and the core RNAP catalytic beta/beta' subunits, correlating with the loading of template DNA into the catalytic cleft of RNAP. It would appear that, for Esigma54 promoters, loading of template DNA within the catalytic cleft of RNAP is dependent on fast ATP hydrolysis steps that trigger changes in the beta' jaw domain, thereby allowing acquisition of the open complex status.
Assuntos
Desnaturação de Ácido Nucleico , RNA Polimerase Sigma 54/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Sequência de Bases , DNA Bacteriano/metabolismo , Escherichia coli , Proteínas de Escherichia coli/metabolismo , Hidrólise , Klebsiella pneumoniae , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Conformação de Ácido Nucleico , Conformação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/metabolismo , RNA Polimerase Sigma 54/genética , Transativadores/metabolismo , Transcrição GênicaRESUMO
DNA opening for transcription-competent open promoter complex (OC) formation by the bacterial RNA polymerase (RNAP) relies upon a complex network of interactions between the structurally conserved and flexible modules of the catalytic beta and beta'-subunits, RNAP-associated sigma-subunit, and the DNA. Here, we show that one such module, the beta'-jaw, functions to stabilize the OC. In OCs formed by the major sigma70-RNAP, the stabilizing role of the beta'-jaw is not restricted to any particular melted DNA segment. In contrast, in OCs formed by the major variant sigma54-RNAP, the beta'-jaw and a conserved sigma54 regulatory domain co-operate to stabilize the melted DNA segment immediately upstream of the transcription start site. Clearly, regulated communication between the mobile modules of the RNAP and the functional domain(s) of the sigma subunit is required for stable DNA opening.
Assuntos
RNA Polimerases Dirigidas por DNA/química , DNA/química , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Thermus/enzimologia , Bacteriófago T7/metabolismo , Sítios de Ligação , Reagentes de Ligações Cruzadas/farmacologia , DNA Bacteriano/genética , Desoxirribonuclease I/metabolismo , Escherichia coli/enzimologia , Heparina/química , Modelos Moleculares , Mutação , Plasmídeos/metabolismo , Permanganato de Potássio/farmacologia , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Fatores de Tempo , Transcrição GênicaRESUMO
We used bacteriophage T7-encoded transcription inhibitor gene protein 2 (gp2) as a probe to study the contribution of the Escherichia coli RNA polymerase (RNAP) beta' subunit jaw domain--the site of gp2 binding--to activator and ATP hydrolysis-dependent open complex formation by the sigma(54)-RNAP. We show that, unlike sigma(70)-dependent transcription, activated transcription by sigma(54)-RNAP is resistant to gp2. In contrast, activator and ATP hydrolysis-independent transcription by sigma(54)-RNAP is highly sensitive to gp2. We provide evidence that an activator- and ATP hydrolysis-dependent conformational change involving the beta' jaw domain and promoter DNA is the basis for gp2-resistant transcription by sigma(54)-RNAP. Our results establish that accessory factors bound to the upstream face of the RNAP, communicate with the beta' jaw domain, and that such communication is subjected to regulation.
Assuntos
RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Substâncias Macromoleculares , Modelos Moleculares , Desnaturação de Ácido Nucleico , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica , Subunidades Proteicas/genética , RNA Polimerase Sigma 54 , Proteínas Repressoras/metabolismo , Fator sigma/genética , Fator sigma/metabolismo , Transcrição GênicaRESUMO
Transcriptional activator proteins that act upon the sigma54-containing form of the bacterial RNA polymerase belong to the extensive AAA+ superfamily of ATPases, members of which are found in all three kingdoms of life and function in diverse cellular processes, often via chaperone-like activities. Formation and collapse of the transition state of ATP for hydrolysis appears to engender the interaction of the activator proteins with sigma54 and leads to the protein structural transitions needed for RNA polymerase to isomerize and engage with the DNA template strand. The common oligomeric structures of AAA+ proteins and the creation of the active site for ATP hydrolysis between protomers suggest that the critical changes in protomer structure required for productive interactions with sigma54-holoenzyme occur as a consequence of sensing the state of the gamma-phosphate of ATP. Depending upon the form of nucleotide bound, different functional states of the activator are created that have distinct substrate and chaperone-like binding activities. In particular, interprotomer ATP interactions rely upon the use of an arginine finger, a situation reminiscent of GTPase-activating proteins.