GENERAL TRANSCRIPTION FACTORS IN YEAST

本应用程序的长期目标是了解更多有关 真核生物转录启动的基本方面。这 过程在所有真核生物中都是高度保守的，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， 控制TATA结合活性的酿酒酵母 蛋白质(TBP)。TBP是一种高度保守的蛋白质，在 真核生物，这对这三种生物的转录启动都是必不可少的 核糖核酸聚合酶。本应用的研究主要集中在 RNA聚合酶的转录启动II.基因的组合 生化研究表明，两种酵母转录因子， Mot1p和Spt3p分别作为负调节因子和正调节因子 TBP活动。Mot1p是一个基本功能，可以从其 正常的DNA结合部位，一个塔塔盒子。Spt3p是一个非本质函数； 其活性可能是中和特定启动子上的Mot1p活性， 从而赋予TBP与DNA结合的启动子专一性。 拟议的实验将使用遗传学、分子和生物化学。 分析Spt3p及其与Mot1p关系的方法和研究 这些转录因子和其他相关转录因子如何与TBP相互作用 控制转录启动。第一，Spt3p之间的相互作用 Mot1p将通过几种方法进行检查，包括隔离 影响Mot1p-Spt3p相互作用的新的mot1突变，直接的 Mot1p-Spt3p的物理相互作用测试和体外转录 尝试定义这些功能的角色和它们的 转录中的相互作用。第二，Spt3p的其他重要方面 将通过体内足迹Spt3p依赖的启动子来阐明， Spt3p依赖启动子的突变分析及其分离和鉴定 Spt3p显性-负性突变分析。最后，其他函数 将研究与Spt3p有关的三个因素 最近寻找新的SPT基因的突变体，以及通过一种 筛选与Mot1p相互作用的其他类似Spt3p的因子。 这些研究应该揭示转录调控的重要方面。 放在酵母里。考虑到酵母和人类之间的保守性，结果是 这些研究的结果将适用于人类的转录。这些 研究与人类疾病相关，因为基因转录改变了 人类与癌症等疾病有牵连。

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.