The proteomes of transcription factories containing RNA polymerases I, II or III.

The proteomes of transcription factories containing RNA polymerases I, II or III.
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DOI:
10.1038/nmeth.1705
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发表时间:
2011-09-25
期刊:
影响因子:
48
通讯作者:
Cook, Peter R.
Cook, Peter R.
中科院分区:
生物学1区
文献类型:
--
作者:
Melnik, Svitlana;Deng, Binwei;Papantonis, Argyris;Baboo, Sabyasachi;Carr, Ian M.;Cook, Peter R.

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人类细胞核含有三种RNA聚合酶(I、II和III),它们转录不同的基因组;这三种聚合酶的活性形式很难分离,因为它们与亚结构结合在一起。在这里,我们描述了从哺乳动物细胞中分离活性RNA聚合酶复合物的纯化方法。分离后,我们通过质谱分析它们的蛋白质含量。每个复合体代表转录工厂核心的一部分;例如,RNA聚合酶II复合体包含RNA聚合酶II特有的亚基以及各种转录因子,但与其他聚合酶复合体共享许多核糖核蛋白;它也富含聚合酶II转录物。我们还描述了一种天然的染色体构象捕获方法,以确认复合物仍然附着在体内发现的相同的DNA模板对。
Human nuclei contain three RNA polymerases (I, II, and III) that transcribe different groups of genes; the active forms of all three are difficult to isolate because they are bound to the substructure. Here, we describe a purification approach for isolating active RNA polymerase complexes from mammalian cells. After isolation, we analyzed their protein content by mass spectrometry. Each complex represents part of the core of a transcription factory; for example, the RNA polymerase II complex contains subunits unique to RNA polymerase II plus various transcription factors, but shares a number of ribonucleoproteins with the other polymerase complexes; it is also rich in polymerase II transcripts. We also describe a native chromosome conformation capture method to confirm that the complexes remain attached to the same pairs of DNA templates found in vivo.
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