STRUCTURE & FUNCTION OF POL III TRANSCRIPTION FACTORS

这个应用程序描述了一个强大的遗传策略的使用 靶向转录因子和辅助因子的突变， RNA聚合酶111转录装置的组成部分 酵母 这种遗传学方法避免了 转录因子的生化纯化，TFIIIB 和TFIIIC，并提供了一个直接的途径，以他们的基因。 的 克隆这些因子的基因并确定 它们的初级结构是这项工作的最初目标。 随后的研究将确定功能重要性的网站， 克隆因子 特别令人感兴趣的是网站， 参与蛋白质-蛋白质和蛋白质-核酸的相互作用 与转录机器的其他组成部分。 最后， 描述了克隆的转录因子 基因用于产生抗体探针和底物， 研究转录因子的翻译后调控 活动 这项工作的长期目标是（i）获得 转录过程的完整分子描述 （二）了解机制; 负责调节转录活性，因此， 控制靶基因的表达。 这个系统可以作为一个模型 为高等真核生物，并提供了结合的优势 基因、分子生物学和生物化学技术， 基因表达中的主要事件，即， 核糖核酸 介绍了基于一个共同主题的几种遗传战略： tRNA无义抑制基因的独特串联排列是 描述了其中下游（supS 1）基因的表达是 依赖于转录的内部启动子， 上游（sup 9-e）基因。 不同的启动子突变， 阻止supS 1表达，从而提供选择， 抑制转录缺陷的基因外突变。 已经分离出突变菌株，其抑制了 A区（sup 9-e A19）启动子突变。 基因特征， 描述了互补分析和基因克隆方案。 用全细胞提取物和 从突变株分离的转录组分 提出作为一种手段，以确定突变的因素，并研究其 作用机制。 转录因子结构的研究- 转录因子的功能关系及调控 活动将使用这些体外系统一起进行 用体内和体外诱变策略。

This application describes the use of a powerful genetic strategy to target mutations in transcription factors and ancillary components of the RNA polymerase 111 transcription apparatus of yeast. This genetic approach circumvents problems encountered with the biochemical purification of the transcription factors, TFIIIB and TFIIIC, and provides a direct route to their genes. The cloning of the genes for these factors and the determination of their primary structures is the initial goal of this work. Subsequent studies will identify sites of functional importance in the cloned factors. Of particular interest are sites that are engaged in protein-protein and protein-nucleic acid interactions with other components of the transcription machinery. Finally, studies are described in which the cloned transcription factor genes are used to generate antibody probes and substrates to investigate the posttramlational regulation of transcription factor activity. The long term objectives of this work are (i) to obtain a complete molecular description of the process of transcription initiation and (ii) to gain an understanding of the mechanisms responsible for regulating transcriptional activity and hence, control of target gene expression. This system serves as a model for higher eukaryotes and offers the advantages of combined genetic, molecular biological and biochemical technologies to study the primary event in gene expression, namely, the transcription of RNA. Several genetic strategies, based on a common theme, are presented: A unique tandem arrangement of tRNA nonsense suppressor genes is described in which expression of a downstream (supS1) gene is dependent upon transcription directed by the internal promoters of an upstream (sup9-e) gene. Different promoter mutations in latter prevent supS1 expression and thus provide a selection for extragenic mutations that suppress the transcriptional defect. Mutant strains have been isolated that suppress the effect of an A-block (sup9-e A19) promoter mutation. Genetic characterization, complementation analysis and a gene cloning protocol are described. In vitro transcription experiments with whole-cell extracts and fractionated transcription components from the mutant strains are proposed as a means to identify the mutant factors and study their mechanism of action. Studies on transcription factor structure- function relationships and the regulation of transcription factor activity will be conducted using these in vitro systems together with in vivo and in vitro mutagenesis strategies.