In vitro analysis of RNA polymerase II elongation complex dynamics
In vitro analysis of RNA polymerase II elongation complex dynamics
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RNA聚合酶II延伸复合物（ECs）组装从核提取物上固定的DNA模板和定量质谱分析。时程实验表明，起始因子TFIIF可以保持与早期EC的结合，而核心延伸因子Spt 4-Spt 5、Paf 1C、Spt 6-Spn 1和Elf 1的水平保持稳定。重要的是，Rpb 1 C-末端结构域（CTD）的动态磷酸化模式以及识别它们的因子作为起始后时间的函数而变化，而不是距离延长。化学抑制Kin 28/Cdk 7阻断丝氨酸5和丝氨酸2磷酸化，影响起始位点的选择，并抑制延伸效率。依赖于CTD磷酸化的EC组分包括加帽酶、帽结合复合物、Set 2和PAF 1复合物。通过概括体内延伸的许多已知特征，该系统揭示了新的细节，阐明了EC相关的核心延伸因子，染色质调节因子和RNA加工因子在转录的每个步骤中如何变化。
RNA polymerase II elongation complexes (ECs) were assembled from nuclear extract on immobilized DNA templates and analyzed by quantitative mass spectrometry. Time course experiments showed that initiation factor TFIIF can remain bound to early ECs, while levels of core elongation factors Spt4-Spt5, Paf1C, Spt6-Spn1, and Elf1 levels remain steady. Importantly, the dynamic phosphorylation patterns of the Rpb1 C-terminal domain (CTD), and the factors that recognize them, change as a function of post-initiation time, rather than distance elongated. Chemical inhibition of Kin28/Cdk7 blocks both Serine 5 and Serine 2 phosphorylation, affects initiation site choice, and inhibits elongation efficiency. EC components dependent on CTD phosphorylation include capping enzyme, Cap Binding Complex, Set2, and the PAF1 complex. By recapitulating many known features of in vivo elongation, this system reveals new details that clarify how EC-associated core elongation factors, chromatin regulators, and RNA processing factors change at each step of transcription.