RESUMO
The transition from transcription initiation to elongation is highly regulated in human cells but remains incompletely understood at the structural level. In particular, it is unclear how interactions between RNA polymerase II (RNA Pol II) and initiation factors are broken to enable promoter escape. Here, we reconstitute RNA Pol II promoter escape in vitro and determine high-resolution structures of initially transcribing complexes containing 8-, 10-, and 12-nt ordered RNAs and two elongation complexes containing 14-nt RNAs. We suggest that promoter escape occurs in three major steps. First, the growing RNA displaces the B-reader element of the initiation factor TFIIB without evicting TFIIB. Second, the rewinding of the transcription bubble coincides with the eviction of TFIIA, TFIIB, and TBP. Third, the binding of DSIF and NELF facilitates TFIIE and TFIIH dissociation, establishing the paused elongation complex. This three-step model for promoter escape fills a gap in our understanding of the initiation-elongation transition of RNA Pol II transcription.
Assuntos
Fosfoproteínas , Regiões Promotoras Genéticas , RNA Polimerase II , Proteína de Ligação a TATA-Box , Fator de Transcrição TFIIB , Fatores de Transcrição , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Humanos , Fator de Transcrição TFIIB/metabolismo , Fator de Transcrição TFIIB/genética , Proteína de Ligação a TATA-Box/metabolismo , Proteína de Ligação a TATA-Box/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Iniciação da Transcrição Genética , Fator de Transcrição TFIIH/metabolismo , Fator de Transcrição TFIIH/genética , Fator de Transcrição TFIIH/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Ligação Proteica , Fator de Transcrição TFIIA/metabolismo , Fator de Transcrição TFIIA/genética , Transcrição Gênica , Elongação da Transcrição Genética , RNA/metabolismo , RNA/genética , Fatores de Transcrição TFII/metabolismo , Fatores de Transcrição TFII/genéticaRESUMO
Cyclin-dependent kinase 7 (CDK7), part of the general transcription factor TFIIH, promotes gene transcription by phosphorylating the C-terminal domain of RNA polymerase II (RNA Pol II). Here, we combine rapid CDK7 kinase inhibition with multi-omics analysis to unravel the direct functions of CDK7 in human cells. CDK7 inhibition causes RNA Pol II retention at promoters, leading to decreased RNA Pol II initiation and immediate global downregulation of transcript synthesis. Elongation, termination, and recruitment of co-transcriptional factors are not directly affected. Although RNA Pol II, initiation factors, and Mediator accumulate at promoters, RNA Pol II complexes can also proceed into gene bodies without promoter-proximal pausing while retaining initiation factors and Mediator. Further downstream, RNA Pol II phosphorylation increases and initiation factors and Mediator are released, allowing recruitment of elongation factors and an increase in RNA Pol II elongation velocity. Collectively, CDK7 kinase activity promotes the release of initiation factors and Mediator from RNA Pol II, facilitating RNA Pol II escape from the promoter.
Assuntos
Quinase Ativadora de Quinase Dependente de Ciclina , Quinases Ciclina-Dependentes , Regiões Promotoras Genéticas , RNA Polimerase II , Iniciação da Transcrição Genética , Humanos , Quinases Ciclina-Dependentes/metabolismo , Quinases Ciclina-Dependentes/genética , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Complexo Mediador/metabolismo , Complexo Mediador/genética , Células HeLa , Fator de Transcrição TFIIH/metabolismo , Fator de Transcrição TFIIH/genética , Células HEK293RESUMO
Transcription initiation, the first step in gene expression, has been studied extensively in dilute buffer, a condition which fails to consider the crowded environment in live cells. Recent reports indicate the kinetics of promoter escape is altered in crowded conditions for a consensus bacterial promoter. Here, we use a real-time fluorescence enhancement assay to study the kinetics of unwound bubble formation and promoter escape for three separate promoters. We find that the effect of crowding on transcription initiation is complex, with lower rates of unwound bubble formation, higher rates of promoter escape, and large variations depending on promoter identity. Based on our results, we suggest that altered conditions of crowding inside a live cell can trigger global changes.