STRUCTURE & FUNCTION OF POL III TRANSCRIPTION FACTORS

该应用程序描述了一种强大的遗传策略的使用 靶向转录因子和辅助因子的突变 核糖核酸聚合酶111转录装置的组成 酵母。这种遗传方法绕过了 转录因子TFIIIB的生化纯化 和TFIIIC，并提供了一条直接进入它们基因的途径。这个 这些因子基因的克隆及其活性测定 它们的初级结构是这项工作的初始目标。 随后的研究将确定功能重要性的位置在 克隆因子。特别令人感兴趣的是 参与蛋白质-蛋白质和蛋白质-核酸相互作用 与转录机器的其他组件。最后， 描述了克隆的转录因子的研究 基因被用来产生抗体探针和底物来 研究转录因子的转录后调控 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 启动和(2)了解机制 负责调节转录活动，因此， 控制靶基因的表达。该系统可作为一种模式 为高等真核生物提供了组合的优势 研究遗传学、分子生物学和生化技术 基因表达中的主要事件，即转录 核糖核酸。 基于一个共同的主题，提出了几种遗传策略： TRNA无义抑制基因的独特串联排列是 描述了其中下游(SupS1)基因的表达 依赖于由内部启动子引导的转录 上游(sup9-e)基因。后者中不同的启动子突变 防止supS1表达，从而为以下项提供选择 抑制转录缺陷的基因外突变。 已经分离出了能够抑制一种病毒作用的突变菌株 A-阻断(sup9-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。 全细胞提取液体外转录实验 突变菌株的分级转录成分是 提出作为一种手段来识别突变因素并研究其 作用机制。转录因子结构的研究 转录因子的功能关系与调控 活动将使用这些体外系统一起进行。 采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.