Experimental analysis of hFACT action during Pol II transcription in vitro.
Experimental analysis of hFACT action during Pol II transcription in vitro.
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DOI：
10.1007/978-1-4939-2392-2_19
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发表时间：
2015-01-01
期刊：
影响因子：
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通讯作者：
Studitsky, Vasily M
中科院分区：
文献类型：
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作者：
Hsieh, Fu-Kai;Kulaeva, Olga I;Studitsky, Vasily M
FACT(促进染色质转录)是一种组蛋白伴侣蛋白，通过染色质促进转录，并在转录过程中促进组蛋白恢复。在这里，我们描述了一个高度纯化的实验系统，它概括了转录染色质的许多重要性质，以及hFACT在体外这一过程中的关键作用。我们介绍了如何制备不同形式的核小体的方案，包括完整的核小体、共价结合的核小体、缺失两个H_2A/2B二聚体之一的核小体(六聚体)和四小体(缺失H_2A/2B两个二聚体的核小体)。这些复合体允许对FACT功能的各个方面进行分析。这些方法和下文描述的其他方法也可以应用于其他染色质改构体和染色质靶向因子的研究。
FACT (facilitates chromatin transcription) is a histone chaperone that facilitates transcription through chromatin and promotes histone recovery during transcription. Here, we describe a highly purified experimental system that recapitulates many important properties of transcribed chromatin and the key aspects of hFACT action during this process in vitro. We present the protocols describing how to prepare different forms of nucleosomes, including intact nucleosome, covalently conjugated nucleosome, nucleosome missing one of the two H2A/2B dimers (hexasome) and tetrasome (a nucleosome missing both H2A/2B dimers). These complexes allow analysis of various aspects of FACT's function. These approaches and other methods described below can also be applied to the study of other chromatin remodelers and chromatin-targeted factors.