TAFS AND GENERAL TRANSCRIPTION FACTORS IN YEAST

转录起始装置包括一组通用因子 与启动子DNA和RNA聚合酶II相关的基因 入会仪式。通用转录因子TFIID是第一个 在组装起始复合体的过程中与启动子DNA结合。这个 天然形式的通用转录因子TFIID由TATA组成 结合蛋白(TBP)及多种TBP相关因子(TAF) 真核生物，我们最近发现，这种情况也适用于 酵母。这项提议旨在识别和克隆人类基因组的基因 全套酿酒酵母TBP相关因子，以及 利用这些基因进行分子遗传学和生物化学实验 旨在阐明酵母TFIID的结构和TAFs的作用 在转录起始阶段。 为了实现这些目标，本提案的三个具体目标是： I)从酿酒酵母中分离编码TBP相关因子的基因 遗传和生化方法；2)研究 体内单个TAF功能的突变；以及3)研究 TAFs和TBP/TAF复合体的体外功能。分离基因 编码TAFs，我们将继续利用一种具有 已经导致了四个TAF基因的分离；四个外源基因 迄今为止研究的RNA聚合酶II CTD截断突变体的抑制子 对TAF进行编码。作为分离TAF基因的生化方法，我们将 鉴定高分子量复合体的蛋白质组分 含有TBP，对这些蛋白质进行微序列分析，并使用 这些信息用于设计用于基因分离的寡核苷酸。至 研究突变对个体体内TAF功能的影响， 我们将构建TAF基因的零突变和条件性突变 分析它们对体内转录的影响。为了调查 TAFs在TFIID复合体、野生型和突变型TFIID中的功能 准备工作将在各种转录检测中进行检验。 旨在评估TAFs在模板组装和合成中的作用 在基础转录和激活转录中。 这项研究的健康相关性源于它对 对控制基因的基本分子机制的理解 表情。

The transcription initiation apparatus includes a set of general factors that associate with promoter DNA and with RNA polymerase II during initiation. The general transcription factor TFIID is the first to associate with promoter DNA during assembly of the initiation complex. The native form of the general transcription factor TFIID consists of the TATA binding protein (TBP) and multiple TBP-associated factors (TAFs) in higher eukaryotes, and we have recently found that this is also the case for yeast. This proposal is designed to identify and clone the genes for the complete set of Saccharomyces cerevisiae TBP-associated factors, and to use these genes to pursue molecular genetic and biochemical experiments designed to elucidate the structure of yeast TFIID and the roles of TAFs in transcription initiation. To accomplish these goals, the three specific aims of this proposal are: i) to isolate genes encoding TBP-associated factors in S. cerevisiae using genetic and biochemical approaches; 2) to investigate the effects of mutations on individual TAF functions in vivo; and 3) to study the function of TAFs and the TBP/TAF complex in vitro. To isolate genes encoding TAFs, we will continue to exploit a genetic selection that has already led to the isolation of four TAF genes; the four extragenic suppressors of RNA polymerase II CTD truncation mutants studied to date encode TAFs. As a biochemical approach to isolating TAF genes, we will identify the protein components of a high molecular weight complex containing TBP, subject these proteins to microsequence analysis, and use this information to design oligonucleotides for gene isolation. To investigate the effects of mutations on individual TAF functions in vivo, we will construct null and conditional mutations in the TAF genes and analyze their effects on transcription in vivo. To investigate the functions of the TAFs in the TFIID complex, wild-type and mutant TFIID preparations will be examined in a variety of transcription assays designed to assess the role of TAFs in template assembly and commitment and in basal and activated transcription. The health relatedness of this research derives from its contribution to the understanding of the basic molecular mechanisms that control gene expression.