STRUCTURE AND FUNCTION OF POL III TRANSCRIPTION FACTORS

本申请中描述的工作利用了遗传和 酵母提供的生物化学优势，以研究一个基本的 在所有真核生物中的重要性，即RNA聚合酶III的转录 (pol III）。 Pol III基因产物参与了许多重要的 细胞过程包括蛋白质合成;前mRNA、前rRNA和 前tRNA成熟;蛋白质转运和端粒合成。 的 pol III基因的表达必须适合于正常执行， 这些过程必须与细胞生长相协调。 长 这项工作的长期目标是获得一个详细的生物化学的理解 pol III基因如何在转录水平上表达和调节， 水平 为此，结合分子遗传学和生物化学 方法被用来调查的性质和功能， 在pol III中起关键作用但目前定义不清的因素 转录。 一种独特的遗传策略已经在四个基因中产生了突变（PCF 1至 PCF 4），每一种都通过pol III增加转录。 PCF1-1 突变影响转录因子（TF）的募集速率 IIIB到DNA。 这一影响将通过时间分辨足迹法进行检验。 还提出了PCF 1的结构-功能研究，其中新的 将分离显性等位基因和条件突变。 后者 将用于通过多拷贝克隆相互作用多肽的基因， 镇压 对PCF 1 -1的研究表明，TFIIIB与DNA的结合是 伴随着翻译后事件，其激活 这个因子，TFIIIB 90。 TFIIIB 90的直接证据 将通过体内标记来寻求后修饰 在纯化和克隆该因子之后进行实验。 我们 目前的转录模型表明，PCF 2 -1可能编码 负责TFIIIB 90活化的催化活性。 的性质和 PCF 2的功能将通过基因测序和转录来确定 问题研究 PCF 4 -1突变鉴定了聚合酶特异性因子， TFIIIB 70，即化学计量限制转录。 实验 被建议检查过表达这种蛋白质的影响， 酵母，并研究其与其他成分的相互作用， 转录机器 最后，一系列的生化和分子 实验概述了测试的假设，PCF 3，一个负 pol III转录的调节因子，介导了对该转录的生长控制。 过程

The work described in this application exploits the genetic and biochemical advantages offered by yeast to study a process of fundamental importance in all eukaryotes, namely transcription by RNA polymerase III (pol III). Pol III gene products participate in a myriad of essential cellular processes including protein syntheses; pre-mRNA, pre-rRNA and pre-tRNA maturation; protein transport and telomere synthesis. The expression of pol III genes must be appropriate for the norma execution of these processes and must be co-ordinated with cell growth. The long term goal of this work is to obtain a detailed biochemical understanding of how pol III genes are expressed and regulated at the transcriptional level. To this end, a combination of molecular genetic and biochemical approaches are being used to investigate the nature and function of factors that play crucial but presently poorly defined roles in pol III transcription. a unique genetic strategy has yielded mutations in four genes (PCF1 to PCF4), each of which increases transcription by pol III. The PCF1-1 mutation influences the rate of recruitment of transcription factor (TF) IIIB to DNA. This effect will be examined by time-resolved footprinting. Structure-function studies of PCF1 are also proposed in which new dominant alleles and conditional mutations will be isolated. The latter will be used to clone genes for interacting polypeptides by multi-copy suppression. Studies on PCF1-1 suggest that the binding of TFIIIB to DNA is accompanied by a posttranslational event which activates a subunit of this factor, TFIIIB90. Direct evidence that TFIIIB90 is posttranslationally modified will be sought by in vivo labeling experiments following the purification and cloning of this factor. Our present transcriptional model suggests that PCF2-1 may encode the catalytic activity responsible for TFIIIB90 activation. The nature and function of PCF2 will be determined by gene sequencing and transcription studies. The PCF4-1 mutation identifies a polymerase specificity factor, TFIIIB70, that is stoichiometrically limiting transcription. Experiments are proposed to examine the effect of over-expressing this protein in yeast and to study its interactions with other components of the transcription machinery. Finally, a series of biochemical and molecular experiments are outlined to test the hypothesis that PCF3, a negative regulator of pol III transcription, mediates growth control over this process.