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首页> 外文期刊>Molecular and Cellular Biology >Proteomics of the Eukaryotic Transcription Machinery: Identification of Proteins Associated with Components of Yeast TFIID by Multidimensional Mass Spectrometry
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Proteomics of the Eukaryotic Transcription Machinery: Identification of Proteins Associated with Components of Yeast TFIID by Multidimensional Mass Spectrometry

机译：真核转录机器的蛋白质组学：通过多维质谱鉴定与酵母TFIID成分相关的蛋白质

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摘要

The general transcription factor TFIID is a multisubunit complex of TATA-binding protein (TBP) and 14 distinct TBP-associated factors (TAFs). Although TFIID constituents are required for transcription initiation of most mRNA encoding genes, the mechanism of TFIID action remains unclear. To gain insight into TFIID function, we sought to generate a proteomic catalogue of proteins specifically interacting with TFIID subunits. Toward this end, TFIID was systematically immunopurified by using polyclonal antibodies directed against each subunit, and the constellation of TBP- and TAF-associated proteins was directly identified by coupled multidimensional liquid chromatography and tandem mass spectrometry. A number of novel protein-protein associations were observed, and several were characterized in detail. These interactions include association between TBP and the RSC chromatin remodeling complex, the TAF17p-dependent association of the Swi6p transactivator protein with TFIID, and the identification of three novel subunits of the SAGA acetyltransferase complex, including a putative ubiquitin-specific protease component. Our results provide important new insights into the mechanisms of mRNA gene transcription and demonstrate the feasibility of constructing a complete proteomic interaction map of the eukaryotic transcription apparatus.
机译：通用转录因子TFIID是TATA结合蛋白（TBP）和14种不同的TBP相关因子（TAF）的多亚基复合物。虽然大多数mRNA编码基因的转录起始需要TFIID成分，但TFIID作用的机制仍不清楚。为了深入了解TFIID功能，我们寻求生成与TFIID亚基特异性相互作用的蛋白质的蛋白质组学目录。为此，使用针对每个亚基的多克隆抗体对TFIID进行了系统免疫纯化，并通过多维液相色谱和串联质谱联用技术直接鉴定了TBP和TAF相关蛋白的构象。观察到许多新颖的蛋白质-蛋白质关联，并详细描述了其中的一些特征。这些相互作用包括TBP与RSC染色质重塑复合体之间的缔合，Swi6p反式激活蛋白与TFIID的TAF17p依赖性缔合以及SAGA乙酰基转移酶复合物的三个新亚基的鉴定，包括推定的泛素特异性蛋白酶组分。我们的结果提供了重要的新见解，对mRNA基因转录的机制，并证明了构建一个完整的蛋白质组相互作用图谱的真核转录仪的可行性。

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