RESUMO
The crystal structure of Thermus aquaticus RNA polymerase holoenzyme (alpha2betabeta'omegasigmaA) complexed with a fork-junction promoter DNA fragment has been determined by fitting high-resolution x-ray structures of individual components into a 6.5-angstrom resolution map. The DNA lies across one face of the holoenzyme, completely outside the RNA polymerase active site channel. All sequence-specific contacts with core promoter elements are mediated by the sigma subunit. A universally conserved tryptophan is ideally positioned to stack on the exposed face of the base pair at the upstream edge of the transcription bubble. Universally conserved basic residues of the sigma subunit provide critical contacts with the DNA phosphate backbone and play a role in directing the melted DNA template strand into the RNA polymerase active site. The structure explains how holoenzyme recognizes promoters containing variably spaced -10 and -35 elements and provides the basis for models of the closed and open promoter complexes.
Assuntos
DNA Bacteriano/química , RNA Polimerases Dirigidas por DNA/química , Regiões Promotoras Genéticas , Fator sigma/química , Thermus/enzimologia , Transcrição Gênica , Sequência de Bases , Sítios de Ligação , Cristalização , Cristalografia por Raios X , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Holoenzimas/química , Holoenzimas/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Subunidades Proteicas , Fator sigma/metabolismo , Moldes GenéticosRESUMO
The sigma subunit is the key regulator of bacterial transcription. Proteolysis of Thermus aquaticus sigma(A), which occurred in situ during crystallization, reveals three domains, sigma(2), sigma(3), and sigma(4), connected by flexible linkers. Crystal structures of each domain were determined, as well as of sigma(4) complexed with -35 element DNA. Exposed surfaces of each domain are important for RNA polymerase binding. Universally conserved residues important for -10 element recognition and melting lie on one face of sigma(2), while residues important for extended -10 recognition lie on sigma(3). Genetic studies correctly predicted that a helix-turn-helix motif in sigma(4) recognizes the -35 element but not the details of the protein-DNA interactions. Positive control mutants in sigma(4) cluster in two regions, positioned to interact with activators bound just upstream or downstream of the -35 element.
Assuntos
Proteínas de Bactérias/química , RNA Polimerases Dirigidas por DNA/química , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Fator sigma/química , Thermus/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Modelos Moleculares , Fator sigma/genética , Fator sigma/metabolismo , Thermus/genética , Transcrição GênicaRESUMO
Cell type-specific transcription during Bacillus sporulation is established by sigmaF. SpoIIAB is an anti-sigma that binds and negatively regulates sigmaF, as well as a serine kinase that phosphorylates and inactivates the anti-anti-sigma SpoIIAA. The crystal structure of sigmaF bound to the SpoIIAB dimer in the low-affinity, ADP form has been determined at 2.9 A resolution. SpoIIAB adopts the GHKL superfamily fold of ATPases and histidine kinases. A domain of sigmaF contacts both SpoIIAB monomers, while 80% of the sigma factor is disordered. The interaction occludes an RNA polymerase binding surface of sigmaF, explaining the SpoIIAB anti-sigma activity. The structure also explains the specificity of SpoIIAB for its target sigma factors and, in combination with genetic and biochemical data, provides insight into the mechanism of SpoIIAA anti-anti-sigma activity.
