Wu S Y, Kershnar E, Chiang C M
Department of Biochemistry, University of Illinois, Urbana 61801, USA.
EMBO J. 1998 Aug 3;17(15):4478-90. doi: 10.1093/emboj/17.15.4478.
TFIID is a multiprotein complex comprised of the TATA-binding protein (TBP) and an array of TBP-associated factors (TAFIIs). Whereas TBP is sufficient for basal transcription in conjunction with other general transcription factors and RNA polymerase II, TAFIIs are additionally required for activator-dependent transcription in mammalian cell-free transcription systems. However, recent in vivo studies carried out in yeast suggest that TAFIIs are not globally required for activator function. The discrepancy between in vivo yeast studies and in vitro mammalian cell-free systems remains to be resolved. In this study, we describe a mammalian cell-free transcription system reconstituted with only recombinant proteins and epitope-tagged multiprotein complexes. Transcriptional activation can be recapitulated in this highly purified in vitro transcription system in the absence of TAFIIs. This TBP-mediated activation is not induced by human mediator, another transcriptional coactivator complex potentially implicated in activator response. In contrast, general transcription factors TFIIH and TFIIA play a significant role in TBP-mediated activation, which can be detected in vitro with Gal4 fusion proteins containing various transcriptional activation domains. Our data, therefore, suggest that TFIIH and TFIIA can mediate activator function in the absence of TAFIIs.
TFIID是一种多蛋白复合物，由TATA结合蛋白（TBP）和一系列TBP相关因子（TAFIIs）组成。虽然TBP与其他通用转录因子和RNA聚合酶II结合足以进行基础转录，但在哺乳动物无细胞转录系统中，激活因子依赖性转录还需要TAFIIs。然而，最近在酵母中进行的体内研究表明，TAFIIs并非激活因子功能所必需。酵母体内研究与体外哺乳动物无细胞系统之间的差异仍有待解决。在本研究中，我们描述了一种仅用重组蛋白和表位标记的多蛋白复合物重建的哺乳动物无细胞转录系统。在没有TAFIIs的情况下，这种高度纯化的体外转录系统可以重现转录激活。这种TBP介导的激活不是由人中介体诱导的，人中介体是另一种可能与激活因子反应有关的转录共激活复合物。相反，通用转录因子TFIIH和TFIIA在TBP介导的激活中起重要作用，这可以在体外通过含有各种转录激活结构域的Gal4融合蛋白检测到。因此，我们的数据表明，在没有TAFIIs的情况下，TFIIH和TFIIA可以介导激活因子功能。
