STRUCTURE/FUNCTION OF POL III TRANSCRIPTION FACTORS

RNA聚合酶III(PolIII)的转录是基本的 在所有真核生物中的重要性，因为它的产物参与了各种 基本的细胞过程，包括蛋白质合成，RNA 加工和蛋白质运输。这项工作的长期目标是 为了获得对以下限制步骤的详细生物化学了解 POL III基因转录。为此，我们正在研究增加的 与“激活”相关的转录：显性和隐性 酿酒酵母PCF1基因突变的研究PCF1编码 TFIIIC含四肽重复(TPR)亚单位 (TFIIIC131)，并负责招募异三聚体 启动因子TFIIIB在DNA转录起始上游的作用 地点。TFIIIB的募集是通过直接的蛋白质-蛋白质来实现的 TFIIIC131与TFIIB相关因子TFIIIB70的相互作用 显性和隐性PCF1突变被认为是促进 TFIIIB组装中的不同部分限制步骤 影响TFIIIC131和/或蛋白质构象变化的研究 TFIIIB70。利用纯化酵母因子和酵母菌进行生化实验 重组蛋白质和分子遗传策略被提出 (1)查明受影响的步骤和“激活”的机制 显性和隐性PCF1突变，(Ii)定义相互作用 TFIIIC131和IFIIIB70之间的结构域和(Iii)评估TPR的作用 结构/功能关系，包括“远程”TPR-TPR TFIIIB复合体组装中的相互作用。这些研究的结果 将为理解转录控制是如何进行的 POL III基因的表达可能在细胞周期中或在 对细胞生长速度或其他化学、生物或 环境因素。此外，调查结果将确定 考虑不同TPR的功能和调节的模式 蛋白质家族。

Transcription by RNA polymerase III (pol III) is of fundamental importance in all eukaryotes since its products participate in a variety of essential cellular processes including protein synthesis, RNA processing and protein transport. The long-term goal of this work is to obtain a detailed biochemical understanding of the limiting steps in pol III gene transcription. To this end, we are studying the increased transcription associated with "activating" dominant and recessive mutations in the PCF1 gene of Saccharomyces cerevisiae. PCF1 encodes the tetratricopeptide repeat (TPR)-containing subunit of TFIIIC (TFIIIC131) and is responsible for recruiting the heterotrimeric initiation factor TFIIIB to the DNA upstream of the transcription start site. The recruitment of TFIIIB is achieved by direct protein-protein interactions between TFIIIC131 and the TFIIB-related factor, TFIIIB70. The dominant and the recessive PCF1 mutations are thought to facilitate different partially limiting steps in the assembly of TFIIIB by affecting distinct protein conformational changes in TFIIIC131 and/or TFIIIB70. Biochemical experiments using purified yeast factors and recombinant proteins and molecular genetic strategies are proposed to (i) identify the steps affected and the mechanisms of "activation" by the dominant and recessive PCF1 mutations, (ii) define the interaction domains between TFIIIC131 and IFIIIB70 and (iii) assess the role of TPR structure/function relationships including "long range" TPR-TPR interactions in TFIIIB complex assembly. The results of these studies will provide a basis for understanding how transcriptional control of pol III gene expression may be achieved during the cell cycle or in response to cell growth rate or other chemical, biological or environmental factors. In addition, the findings will establish aparadigm for considering the function and regulation of the diverse TPR family of proteins.