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体外分析 RNA 聚合酶 II 延伸复合物的动态变化。

In vitro analysis of RNA polymerase II elongation complex dynamics.

机构信息

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.

Department of Cancer Biology, Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.

出版信息

Genes Dev. 2019 May 1;33(9-10):578-589. doi: 10.1101/gad.324202.119. Epub 2019 Mar 7.

DOI:10.1101/gad.324202.119
PMID:30846429
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6499329/
Abstract

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 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 postinitiation time rather than distance elongated. Chemical inhibition of Kin28/Cdk7 in vitro blocks both Ser5 and Ser2 phosphorylation, affects initiation site choice, and inhibits elongation efficiency. EC components dependent on CTD phosphorylation include capping enzyme, cap-binding complex, Set2, and the polymerase-associated factor (PAF1) complex. By recapitulating many known features of in vivo elongation, this system reveals new details that clarify how EC-associated factors change at each step of transcription.

摘要

RNA 聚合酶 II 延伸复合物（EC）在固定化 DNA 模板上从核提取物中组装，并通过定量质谱分析。时程实验表明，起始因子 TFIIF 可以与早期 EC 结合，而核心延伸因子 Spt4-Spt5、Paf1C、Spt6-Spn1 和 Elf1 的水平保持稳定。重要的是，Rpb1 C 末端结构域（CTD）的动态磷酸化模式以及识别它们的因子随着起始后时间的变化而变化，而不是随着延伸距离的变化而变化。体外抑制激酶 Kin28/Cdk7 可阻断 Ser5 和 Ser2 磷酸化，影响起始位点选择，并抑制延伸效率。依赖 CTD 磷酸化的 EC 成分包括加帽酶、帽结合复合物、Set2 和聚合酶相关因子（PAF1）复合物。通过再现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 相关因子在转录的每个步骤如何变化。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/79af/6499329/ae2813adc936/578f06.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/79af/6499329/d502b41e6e23/578f01.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/79af/6499329/469137e3aa05/578f02.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/79af/6499329/fde22551b0b5/578f05.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/79af/6499329/ae2813adc936/578f06.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/79af/6499329/d502b41e6e23/578f01.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/79af/6499329/469137e3aa05/578f02.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/79af/6499329/df4263bb1069/578f03.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/79af/6499329/12bf372b8b3b/578f04.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/79af/6499329/fde22551b0b5/578f05.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/79af/6499329/ae2813adc936/578f06.jpg

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