RESUMO
The phage shock protein (Psp) response maintains integrity of the inner membrane (IM) in response to extracytoplasmic stress conditions and is widely distributed amongst enterobacteria. Its central component PspA, a member of the IM30 peripheral membrane protein family, acts as a major effector of the system through its direct association with the IM. Under non-stress conditions PspA also negatively regulates its own expression via direct interaction with the AAA+ ATPase PspF. PspA has a counterpart in cyanobacteria called Vipp1, which is implicated in protection of the thylakoid membranes. PspA's and Vipp1's conserved N-terminal regions contain a putative amphipathic helix a (AHa) required for membrane binding. An adjacent amphipathic helix b (AHb) in PspA is required for imposing negative control upon PspF. Here, purified peptides derived from the putative AH regions of PspA and Vipp1 were used to directly probe their effector and regulatory functions. We observed direct membrane-binding of AHa derived peptides and an accompanying change in secondary structure from unstructured to alpha-helical establishing them as bona fide membrane-sensing AH's. The peptide-binding specificities and their effects on membrane stability depend on membrane anionic lipid content and stored curvature elastic stress, in agreement with full length PspA and Vipp1 protein functionalities. AHb of PspA inhibited the ATPase activity of PspF demonstrating its direct regulatory role. These findings provide new insight into the membrane binding and function of PspA and Vipp1 and establish that synthetic peptides can be used to probe the structure-function of the IM30 protein family.
Assuntos
Proteínas de Bactérias/química , Proteínas de Escherichia coli/química , Proteínas de Choque Térmico/química , Proteínas de Membrana/química , Peptídeos/química , Fosfatidilcolinas/química , Fosfatidilgliceróis/química , Transativadores/química , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Membrana/metabolismo , Peptídeos/síntese química , Fosfatidilcolinas/metabolismo , Fosfatidilgliceróis/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estresse Fisiológico , Synechocystis/química , Synechocystis/metabolismo , Transativadores/metabolismo , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismoRESUMO
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.