STRUCTURAL & FUNCTION OF POL III TRANSCRIPTION FACTORS

描述（由申请人提供）RNA聚合酶（pol）III的转录在所有真核生物中具有根本的重要性，因为其产物，包括5S RNA、tRNA、U6 snRNA、RNase P RNA和7SL RNA，对于蛋白质合成、RNA加工、蛋白质转运和其它细胞过程是必需的。pol III基因的转录与细胞生长紧密耦合，并与pol I的大核糖体RNA的转录共调节。这些转录过程约占生长细胞核转录的80%，它们的协调调节被认为对代谢经济和生物适应性很重要。构成pol III转录机制的大多数蛋白质从酵母到人都是保守的。因此，在几乎所有情况下，从酵母中的pol III转录研究中获得的知识很容易翻译到人类细胞中。在高等真核生物中，pol III的转录被肿瘤抑制基因（p53和RB）的突变以及病毒和细胞癌基因的细胞转化激活。因此，认为转化细胞的生长速率升高部分依赖于其高水平的pol III转录。 在各种条件下受调控的pol III转录组分的身份及其调控机制在任何系统中都没有明确定义。因此，本研究的长期目标是确定pol III系统中的调节目标，并确定控制其功能的方法。为此，本申请中的实验将提供对pol III起始因子TFIIIB在酿酒酵母中协同组装中的限制性步骤的详细生物化学理解，即TFIIIC的含有三肽重复序列（TPR）的亚基（TFIIIC 131）和TFIIIB的TFIIIB相关亚基（Brf 1）之间的相互作用。这项研究也将作为一个有价值的范例，了解结合特异性和功能的普遍存在的TPR基序在组装的多亚基复合物。此外，实验将确定pol III因子，其响应上游信号通路并介导酵母中的转录抑制。这些因素将使用生物化学和分子遗传学方法进行检查，以阐明其调节的分子机制。将通过体内和体外试验的组合明确检验这些研究过程中开发的假设。

DESCRIPTION (provided by applicant) Transcription by RNA polymerase (pol) III is of fundamental importance in all eukaryotes since its products, which include 5S RNA, tRNA, U6 snRNA, RNase P RNA and 7SL RNA, are essential for protein synthesis, RNA processing, protein transport and other cellular processes. The transcription of pol III genes is tightly coupled with cell growth and is co-regulated with transcription of the large ribosomal RNAs by pol I. These transcriptional processes account for about 80% of nuclear transcription in growing cells and their coordinate regulation is thought to be important for metabolic economy and biological fitness. The majority of the proteins that comprise the pol Ill transcription machinery are conserved from yeast to humans. Thus, in almost all cases, the knowledge obtained from studies on pol III transcription in yeast is readily translated into human cells. In higher eukaryotes, pol III transcription is activated by mutations in tumor suppressor genes (p53 and RB) and by cell transformation with viral and cellular oncogenes. It is thought, therefore, that the elevated growth rate of transformed cells is dependent, in part, on their high levels of pol III transcription. The identities of the pol III transcription components that are subject to regulation under a variety of conditions and the mechanisms of their regulation are not well-defined in any system. Accordingly, the long-term objectives of this research are to identify the regulatory targets in the pol III system and determine the ways in which their function is controlled. To this end, the experiments in this application will provide a detailed biochemical understanding of a limiting step in the concerted assembly of the pol III initiation factor TFIIIB in Saccharomyces cerevisiae, namely the interaction between the tetratricopeptide repeat (TPR)- containing subunit of TFIIIC (TFIIIC131) and TFIIB-related subunit of TFIIIB (Brf1). This study will also serve as a valuable paradigm for understanding the binding specificity and function of the ubiquitous TPR motif in the assembly of multi-subunit complexes. In addition, the experiments will identify the pol III factors that respond to upstream signaling pathways and mediate transcriptional repression in yeast. These factors will be examined using biochemical and molecular genetic methods to elucidate the molecular mechanisms of their regulation. The hypotheses developed in course of these studies will be explicitly tested by a combination of in vivo and in vitro assays.