Ozer J, Bolden A H, Lieberman P M
Wistar Institute, Philadelphia Pennsylvania 19104, USA.
J Biol Chem. 1996 May 10;271(19):11182-90. doi: 10.1074/jbc.271.19.11182.
The general transcription factor IIA (TFIIA) binds to the TATA binding protein (TBP) and mediates transcriptional activation by distinct classes of activators. To elucidate the function of TFIIA in transcriptional activation, point mutants were created in the human TFIIA-gamma subunit at positions conserved with the yeast homologue. We have identified a class of TFIIA mutants that stimulate TBP-DNA binding (T-A complex) but fail to support transcriptional activation by several different activators, suggesting that these mutants are defective in their ability to facilitate an activation step subsequent to TBP promoter binding. Point mutations of the hydrophobic core of conserved residues from 65 to 74 resulted in various activation-defective phenotypes. These residues were found to be important for TFIIA gamma-gamma interactions, suggesting that gamma-gamma interactions are critical for TFIIA function as a coactivator. A subset of these TFIIA-gamma mutations disrupted transcriptional activation by all activators tested, except for the Epstein-Barr virus-encoded Zta protein. The gamma Y65F, gamma W72A, and gamma W72F mutants mediate Zta activation, but not GAL4-AH, AP-1, GAL4-CTF, or GAL4-VP16 activation. The gamma W72A mutant failed to stimulate TFIID-DNA binding (D-A complex) but was able to form a complex with TFIID and DNA in the presence of Zta (Z-D-A complex). Thus, the ability of Zta to activate transcription with gamma W72A appears to result from a unique ability to form the stable Z-D-A complex with this mutant. Our results show that different activators utilize the general factor TFIIA in unique ways and that TFIIA contributes transcription activation functions in addition to the facilitation of TBP-DNA binding.
通用转录因子IIA（TFIIA）与TATA结合蛋白（TBP）结合，并介导不同类型激活因子的转录激活。为了阐明TFIIA在转录激活中的功能，在人TFIIA-γ亚基中与酵母同源物保守的位置创建了点突变体。我们鉴定出一类TFIIA突变体，它们能刺激TBP与DNA的结合（T-A复合物），但不能支持几种不同激活因子的转录激活，这表明这些突变体在促进TBP与启动子结合后的激活步骤方面存在缺陷。保守残基65至74的疏水核心的点突变导致了各种激活缺陷表型。发现这些残基对TFIIAγ-γ相互作用很重要，这表明γ-γ相互作用对于TFIIA作为共激活因子的功能至关重要。这些TFIIA-γ突变体的一个子集破坏了所有测试激活因子的转录激活，但不包括爱泼斯坦-巴尔病毒编码的Zta蛋白。γY65F、γW72A和γW72F突变体介导Zta激活，但不介导GAL4-AH、AP-1、GAL4-CTF或GAL4-VP16激活。γW72A突变体不能刺激TFIID与DNA的结合（D-A复合物），但在存在Zta的情况下能够与TFIID和DNA形成复合物（Z-D-A复合物）。因此，Zta与γW72A一起激活转录的能力似乎源于与该突变体形成稳定Z-D-A复合物的独特能力。我们的结果表明，不同的激活因子以独特的方式利用通用因子TFIIA，并且TFIIA除了促进TBP与DNA的结合外，还具有转录激活功能。
