RESUMO
TFIIE and the archaeal homolog TFE enhance DNA strand separation of eukaryotic RNAPII and the archaeal RNAP during transcription initiation by an unknown mechanism. We have developed a fluorescently labeled recombinant M. jannaschii RNAP system to probe the archaeal transcription initiation complex, consisting of promoter DNA, TBP, TFB, TFE, and RNAP. We have localized the position of the TFE winged helix (WH) and Zinc ribbon (ZR) domains on the RNAP using single-molecule FRET. The interaction sites of the TFE WH domain and the transcription elongation factor Spt4/5 overlap, and both factors compete for RNAP binding. Binding of Spt4/5 to RNAP represses promoter-directed transcription in the absence of TFE, which alleviates this effect by displacing Spt4/5 from RNAP. During elongation, Spt4/5 can displace TFE from the RNAP elongation complex and stimulate processivity. Our results identify the RNAP "clamp" region as a regulatory hot spot for both transcription initiation and transcription elongation.
Assuntos
Proteínas Arqueais/química , Proteínas Cromossômicas não Histona/química , RNA Polimerases Dirigidas por DNA/metabolismo , Transcrição Gênica , Fatores de Elongação da Transcrição/química , Sequência de Aminoácidos , Archaea/genética , Archaea/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Sequência de Bases , Sítios de Ligação , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Transferência Ressonante de Energia de Fluorescência , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Sulfolobus solfataricus/metabolismo , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismoRESUMO
All RNAPs (RNA polymerases) repeatedly make use of their DNA template by progressing through the transcription cycle multiple times. During transcription initiation and elongation, distinct sets of transcription factors associate with multisubunit RNAPs and modulate their nucleic-acid-binding and catalytic properties. Between the initiation and elongation phases of the cycle, the factors have to be exchanged by a largely unknown mechanism. We have shown that the binding sites for initiation and elongation factors are overlapping and that the binding of the factors to RNAP is mutually exclusive. This ensures an efficient exchange or 'swapping' of factors and could furthermore assist RNAP during promoter escape, enabling robust transcription. A similar mechanism applies to the bacterial RNAP system. The elongation factors are evolutionarily conserved between the bacterial (NusG) and archaeo-eukaryotic (Spt5) systems; however, the initiation factors [σ and TBP (TATA-box-binding protein)/TF (transcription factor) B respectively] are not. Therefore we propose that this factor-swapping mechanism, operating in all three domains of life, is the outcome of convergent evolution.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Fatores de Transcrição , RNA Polimerases Dirigidas por DNA/genética , Evolução Molecular , Modelos Moleculares , Regiões Promotoras Genéticas , Biossíntese de Proteínas , Fatores de Transcrição/metabolismoRESUMO
The complex organization of the transcription machinery has been revealed mainly by biochemical and crystallographic studies. X-ray structures describe RNA polymerases and transcription complexes on an atomic level, but fail to portray their dynamic nature. The use of fluorescence techniques has made it possible to add a new layer of information to our understanding of transcription by providing details about the structural rearrangement of mobile elements and the network of interactions within transcription complexes in solution and in real-time.