STRUCTURAL & FUNCTION OF POL III TRANSCRIPTION FACTORS

RNA聚合酶（pol） III的转录在所有真核生物中都是至关重要的，因为其产物包括5S RNA、tRNA、U6 snRNA、RNase P RNA和7SL RNA，是蛋白质合成、RNA加工、蛋白质运输和其他细胞过程所必需的。pol III基因的转录与细胞生长紧密耦合，并与pol i对大核糖体rna的转录共同调节。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 - 1转录机制的大多数蛋白质从酵母到人类都是保守的。因此，在几乎所有情况下，从酵母中pol III转录的研究中获得的知识很容易转化为人类细胞。在高等真核生物中，pol III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，人们认为，转化细胞的高生长速度部分依赖于它们高水平的pol III转录。在各种条件下受到调控的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系统中的调控目标，并确定控制其功能的方式。为此，本应用程序中的实验将提供对酵母中pol III起始因子TFIIIB协同组装的限制步骤的详细生化理解，即包含TFIIIC的四肽重复（TFIIIC131）和TFIIIB相关亚基（Brf1）之间的相互作用。该研究也将为理解多亚基复合物组装中普遍存在的TPR基序的结合特异性和功能提供有价值的范例。此外，实验将确定响应上游信号通路和介导酵母转录抑制的pol III因子。这些因素将使用生化和分子遗传学方法来阐明其调控的分子机制。在这些研究过程中提出的假设将通过体内和体外试验的结合进行明确的检验。

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.