The yeast RNA polymerase III transcription machinery:: A paradigm for eukaryotic gene activation
The yeast RNA polymerase III transcription machinery:: A paradigm for eukaryotic gene activation
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DOI：
10.1101/sqb.1998.63.381
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发表时间：
1998-01-01
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通讯作者：
Sentenac, A
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作者：
Chédin, S;Ferri, ML;Sentenac, A
冷泉港定量生物学研讨会，第LXIII卷，© 1998冷泉港实验室出版社0-87969-550-1/98。381 tRNA基因激活。tRNA启动子由两个不同的序列元件A和B区组成，在酵母基因组中鉴定的273个tRNA基因中，这些启动子元件之间的距离可以从31 bp到93 bp广泛变化。因此，我们期望根据DNA足迹特性分离两种不同的DNA结合蛋白。事实并非如此。被称为TFIIIC或Tau的纯化因子是一种非常大的多亚基蛋白，约600 kD，与多种tRNA基因的分裂启动子相互作用（Camier et al. 1985; Gabrielsen et al. 1989）。虽然这两种DNA结合活性不能用常规方法分离，但TFIIIC-DNA复合物的电子显微镜（Schultz et al. 1989）和选择性蛋白水解（Marzouki et al. 1986）证明存在两个连接的DNA结合结构域，τA和τB，每个约300 kD，每个与一个启动子元件相互作用。τB与B嵌段的结合占主导地位，并增强了τA与A嵌段结合的能力。当用一系列具有可变A至B区段距离的伸展或内部缺失的tRNA 3 Leu基因进行攻击时，TFIIICDNA相互作用显示出对可变AB距离及其相对螺旋方向的显著适应性（Baker等，1987; Fabrizio等，1987）。tRNA基因激活的主要步骤是TFIIIC与基因内启动子的结合。TFIIIC的主要功能是在转录起始位点上游组装TFIIIB（Kassavetis et al. 1990）。在研究具有非典型启动子构型的U6 RNA基因时，在终止子信号下游具有远端B阻断，我们发现使用纯化的转录系统不需要TFIIIC，而B阻断以及由此推断的TFIIIC在粗提物中是必需的（Moenne等，1990;盖拉赫等，1995）。我们推测TFIIIC可能具有抵消染色质抑制的额外作用。事实上，TFIIIC可以用预组装的核小体重新激活U6 RNA基因（Burnol et al. 1993）。染色质破坏在体内，诱导组蛋白H4耗尽，刺激各种启动子缺陷基因的转录，但不是野生型U6 RNA基因。因此，在染色质组装和转录复合物形成之间存在竞争，但这种竞争更有利于转录因子（Marsolier et al.1995）。研究TFIIIC与染色质模板的相互作用将是有趣的。我们的初步结果没有给出任何建议，TFIIIC被赋予组蛋白乙酰转移酶活性，至少不是游离组蛋白。值得注意的是，TFIIIC具有不同功能的集合，这些功能被分配给II类基因中的不同蛋白质。它在增强子结合蛋白（在距离起始位点可变距离处的B-阻断相互作用）、近端元件结合因子（通过A-阻断结合）、激活剂（通过TFIIIB结合;见下文）和抗阻遏物（缓解染色质阻遏）中发挥作用。所有这些功能组合成一个灵活的多亚基因子，以实现pol III系统的最佳效率。为了深入研究酵母TFIIIC的结构和功能，我们对TFIIIC进行了鉴定和分子生物学研究，
Cold Spring Harbor Symposia on Quantitative Biology, Volume LXIII.© 1998 Cold Spring Harbor Laboratory Press 0-87969-550-1/98. 381 tRNA gene activation. The tRNA promoters are made of two different sequence elements, the A and B blocks, and in the 273 tRNA genes identified in the yeast genome, the distance between these promoter elements can vary widely from 31 bp to 93 bp. Therefore, we expected to isolate two distinct DNA-binding proteins on the basis of their DNA footprinting properties. This was not the case. The purified factor, called TFIIIC or Tau, was an exceptionally large multisubunit protein of about 600 kD that interacted with the split promoter of a variety of tRNA genes (Camier et al. 1985; Gabrielsen et al. 1989). Although the two DNA-binding activities could not be separated by conventional means, electron microscopy of TFIIIC-DNA complexes (Schultz et al. 1989) and selective proteolysis (Marzouki et al. 1986) demonstrated the existence of two linked DNA-binding domains, τA and τB, of about 300 kD each, each interacting with one promoter element. Binding of τB to the B block is predominant and enhances the ability of τA to bind to the A block. When challenged with a series of stretched or internally deleted tRNA3 Leu genes having variable A to B block distances, the TFIIICDNA interaction displayed a remarkable adaptability to the variable AB distances and to their relative helical orientation (Baker et al. 1987; Fabrizio et al. 1987). The primary step in tRNA gene activation is the binding of TFIIIC to the intragenic promoter. The main function of TFIIIC is then to assemble TFIIIB upstream of the transcription start site (Kassavetis et al. 1990). While studying the U6 RNA gene which has an atypical promoter configuration, with a distal B block downstream from the terminator signal, we found no requirement for TFIIIC using a purified transcription system, whereas the B block, and by inference TFIIIC, was necessary in crude extracts (Moenne et al. 1990; Gerlach et al. 1995). We surmised that TFIIIC may have the additional role of counteracting chromatin repression. Indeed, TFIIIC could reactivate the U6 RNA gene with preassembled nucleosomes (Burnol et al. 1993). Chromatin disruption in vivo, induced by histone H4 depletion, stimulated the transcription of various promoter-deficient genes, but not of wild-type U6 RNA genes. Therefore, there is a competition between chromatin assembly and transcription complex formation, but this competition is much in favor of the transcription factors (Marsolier et al. 1995). It will be interesting to investigate the interaction of TFIIIC with chromatin templates. Our preliminary results give no suggestion that TFIIIC is endowed with histone acetyltransferase activity, at least not for free histones. It is remarkable that TFIIIC possesses an assemblage of different functions that are assigned to separate proteins in class II genes. It plays the part of enhancer-binding proteins (B-block interaction at a variable distance from the start site), of proximal element-binding factors (through A-block binding), of an activator (through TFIIIB binding; see below), and of an antirepressor (relieving chromatin repression). All of these functions are combined into a flexible multisubunit factor for optimal efficiency of the pol III system. To dissect the structure and the function of yeast TFIIIC, we have undertaken the identification and molecular