TRANSCRIPTION FACTOR MUTANTS OF YEAST

拟议研究的长期目标是确定因素 它们在RNA聚合酶的转录启动中起重要作用 II在活体内，并确定这些因素控制的机制 这一过程。这些研究的最初焦点将是塔塔盒子- 结合通用转录因子(TATA盒结合蛋白或TBP)。在… RNA聚合酶II转录的启动子，TBP与TATA盒的结合 启动多蛋白质预起始复合体的有序组装 这足以支持基础水平的转录启动 体外培养。对调控的反应需要额外的蛋白质 信号。由于TBP结合TATA盒是预引发的第一步 复杂的装配、TBP是调控行动的重要靶点 各种因素。这项提案的具体目的是调查三个 TBP功能的不同方面：TBP作为目标的作用(L) 转录激活子；(2)序列特异性和 TBP与DNA的定向结合；(3)两个基因的功能 TBP的结构域可能是相互作用的目标 蛋白质。为了实现这些目标，基因和 将使用生化方法来识别和表征 酵母中与TBP相互作用并调节其活性的蛋白质 酿酒酵母。更确切地说，基因选择将是 以确定抑制或增强表型的突变 由TBP突变体授予。这些研究的基础是不同的 TBP突变体的类别，包括以前已确定的突变体和新的突变体 这将在拟议的研究过程中被隔离。一 现有的一类TBP突变体在激活的转录中表现出缺陷 在活体内，第二类显示DNA结合的变化 专一性。由这些基因引起的改变表型的非连锁突变 将对TBP突变体进行鉴定，并克隆相应的基因 并通过遗传学和分子方法进行研究。其后，至 阐明这些基因产物改变TBP的机制 功能，蛋白质将被提纯并通过生化方法进行分析 这将包括体外转录和DNA结合分析。自.以来 RNA启动转录的蛋白质及其机制 聚合酶II在整个进化过程中高度保守， 从这些酵母研究中学到的信息将显著 促进我们对其他真核生物转录的理解。因此， 拟议中的研究有望为 人类转录启动的调节，其中一个基因 这一过程中的变化可以扰乱正常的细胞生长和 分化，导致癌症等疾病。

The long-term objectives of the proposed research are to identify factors that play an important role in transcription initiation by RNA polymerase II in vivo and to determine the mechanisms by which these factors govern this process. The initial focus of these studies will be the TATA box- binding general transcription factor (TATA box-binding protein or TBP). At promoters transcribed by RNA polymerase II, binding of TBP to the TATA box initiates the ordered assembly of a multi-protein preinitiation complex that is sufficient to support basal levels of transcription initiation in vitro. Additional proteins are required for the response to regulatory signals. Since TATA box binding by TBP is the first step in preinitiation complex assembly, TBP is an important target for the action of regulatory factors. The Specific Aims of this proposal are to investigate three different aspects of TBP function: (l) the role of TBP as a target for transcriptional activators; (2) the determinants of sequence-specific and orientation-specific DNA binding by TBP; and (3) the functions of two structural domains of TBP that are likely targets for interacting proteins. To accomplish these aims, a combination of genetic and biochemical approaches will be employed to identify and characterize proteins that interact with and regulate the activity of TBP in the yeast Saccharomyces cerevisiae. More specifically, genetic selections will be performed to identify mutations that suppress or enhance the phenotypes conferred by TBP mutants. The foundations for these studies are different classes of TBP mutants, both previously identified mutants and new mutants that will be isolated during the course of the proposed research. One existing class of TBP mutants exhibits defects in activated transcription in vivo, and a second class exhibits alterations in DNA-binding specificity. Unlinked mutations that alter the phenotypes caused by these TBP mutants will be characterized, and corresponding genes will be cloned and studied by genetic and molecular approaches. Subsequently, to elucidate the mechanisms by which the products of these genes alter TBP function, proteins will be purified and analyzed by biochemical methods that will include in vitro transcription and DNA-binding assays. Since the proteins and mechanisms employed in transcription initiation by RNA polymerase II have been highly conserved throughout evolution, the information that is learned from these studies in yeast will significantly advance our understanding of transcription in other eukaryotes. Thus, the proposed research is expected to provide valuable insights into the regulation of transcription initiation in humans where a genetic alteration in this process can disrupt normal cell growth and differentiation, leading to cancer and other diseases.