GENERAL TRANSCRIPTION FACTORS IN YEAST

此应用程序的长期目标是了解更多有关 真核生物转录起始的基本方面。 这 这一过程在所有真核生物中都高度保守，从酵母到 人类 这些研究将集中在酵母中发现的蛋白质因子上， 酿酒酵母，其控制TATA结合的活性 蛋白（TBP）。 TBP是一种高度保守的蛋白质， 真核生物，这是必不可少的转录启动所有三个 核RNA聚合酶。 本申请的研究重点是 RNA聚合酶II的转录起始。 基因的组合 生物化学研究表明，两种酵母转录因子， Mot 1 p和Spt 3 p分别作为负调节因子和正调节因子， TBP活性。 Mot 1 p是一个重要的功能，可以从其组织中去除TBP。 一个正常的DNA结合位点，一个TATA盒。 Spt 3 p是一个非必需的功能; 其活性可能是在特定启动子处抵消Mot 1 p活性， 从而赋予TBP与DNA结合的启动子特异性。 拟议中的实验将使用遗传、分子和生物化学 分析Spt 3 p及其与Mot 1 p的关系， 这些和其他相关转录因子如何与TBP相互作用， 控制转录起始。 首先，Spt 3 p之间的相互作用 和Mot 1 p将通过几种方法进行研究，包括分离 影响Mot 1 p-Spt 3 p相互作用的新mot 1突变， Mot 1 p-Spt 3 p物理相互作用和体外转录试验 尝试定义这些功能的作用及其 转录中的相互作用。 第二，Spt 3 p的其他重要方面 将通过Spt 3 p依赖性启动子的体内足迹来阐明， Spt 3 p依赖性启动子的突变分析，以及分离和 Spt 3 p显性阴性突变的分析。 最后，其他功能 将研究与Spt 3 p相关的三个因素，包括在一个 最近寻找新的SPT基因的突变体，以及通过基因组学鉴定的因素。 筛选与Mot 1 p相互作用的其他Spt 3 p样因子。 这些研究应该揭示转录控制的重要方面 在酵母中。 考虑到酵母和人类之间的保守性， 将适用于人类的转录。 这些 这些研究与人类疾病有关，因为基因组中的转录改变， 人类与癌症等疾病有关。

The long-term objectives of this application are to learn more about fundamental aspects of transcription initiation in eukaryotes. This process is strongly conserved in all eukaryotes, ranging from yeast to human. The studies will focus on protein factors discovered in the yeast, Saccharomyces cerevisiae, that control the activity of TATA-binding protein (TBP). TBP is a highly conserved protein, found throughout eukaryotes, that is essential for transcription initiation by all three nuclear RNA polymerases. The studies in this application are focused on transcription initiation by RNA polymerase II. A combination of genetic and biochemical studies have shown that two yeast transcription factors, Mot1p and Spt3p, act as negative and positive regulators, respectively, of TBP activity. Mot1p is an essential function that can remove TBP from its normal DNA binding site, a TATA box. Spt3p is a nonessential function; its activity may be to counteract Mot1p activity at particular promoters, thereby conferring promoter-specificity on TBP binding to DNA. The proposed experiments are to use genetic, molecular, and biochemical approaches to analyze Spt3p and its relationship with Mot1p, and to study how these and other related transcription factors interact with TBP to control transcription initiation. First, the interaction between Spt3p and Mot1p will be examined by several approaches, including the isolation of new mot1 mutations that affect the Mot1p-Spt3p interaction, a direct test for Mot1p-Spt3p physical interaction, and in vitro transcription experiments to attempt to define the roles of these functions and their interactions in transcription. Second, other important aspects of Spt3p will be elucidated by in vivo footprinting of an Spt3p-dependent promoter, mutational analysis of an Spt3p-dependent promoter, and the isolation and analysis of Spt3p dominant-negative mutations. Finally, other functions related to Spt3p will be studied, including three factors identified in a recent mutant hunt for new SPT genes, and factors to be identified by a screen for other Spt3p-like factors that interact with Mot1p. These studies should reveal important aspects of transcriptional control in yeast. Given the conservation between yeast and humans, the results from these studies will be applicable to transcription in humans. These studies are relevant to human disease, since altered transcription in humans has been implicated in diseases such as cancer.