TRANSCRIPTION FACTOR MUTANTS OF YEAST

描述(改编自调查人员的摘要)：长期 拟议研究的目标是确定起重要作用的因素 RNA聚合酶II在体内转录中的作用及确定 这些因素控制这一过程的机制。的中心焦点 这笔赠款是TATA盒结合蛋白(TBP)，它起着至关重要的作用 在所有三种核RNA聚合酶的转录启动过程中。在发起人 由POL II转录，TBP与TATA盒的结合是 一系列事件，最终导致RNA消息的产生。 因此，了解控制TBP的因素是至关重要的一步 了解真核生物中基因表达的调控。这个 特定的目标代表了以前对酵母菌研究的延伸 酿酒酵母TBP。具体目的1是研究RTF1的功能，a 对TBP的TATA位点选择和TBP的TATA位点选择 文字记录延长。RTF1及其相关蛋白将被提纯， RTF1调控的基因将使用DNA微阵列进行鉴定，基因 将进行选择以确定rtf1的基因外抑制物 突变。此外，POL II的磷酸化状态与 随着转录周期的延长，将分析rtf1等突变体 菌株。具体目标2是阐明TBP作为以下目标的重要性 体内转录调节因子。染色质免疫沉淀和 体内足迹方法将被用来确定 基因特异性和全局作用的转录因子在招聘中的作用 TBP为高度调控的模型启动子。具体目标3是分析两个 TBP突变体的不同类别，并获得对两个基本方面的见解 Pol II转录：预起始的定向特异性组装 转录启动的复合体和调控。TBP突变体展示 体内反转的DNA结合极性将使用体外DNA进行分析 裂解和转录检测。在特定情况下改变的TBP突变体 并表现出新的表型，表明存在缺陷 转录调控，将使用生化和遗传两种方法进行研究 接近了。由于蛋白质和机制用于调节 RNA聚合酶II在真核生物中是保守的，这种信息是 从这些酵母研究中学到的东西将极大地促进我们对 在人类的转录过程中，这个过程的中断会导致重要的 人类疾病，包括癌症和艾滋病。

DESCRIPTION (Adapted from the investigator's abstract): The long-term objectives of the proposed research are to identify factors that play important roles in transcription by RNA polymerase II in vivo and to determine the mechanisms by which these factors govern this process. The central focus of this grant is the TATA box-binding protein (TBP), which plays an essential role in transcription initiation by all three nuclear RNA polymerases. At promoters transcribed by Pol II, binding of TBP to the TATA box is the first step in a cascade of events that culminates in the production of an RNA message. Therefore, an understanding of the factors that control TBP is a critical step toward understanding the regulation of gene expression in eukaryotes. The Specific Aims represent extensions of previous studies on Saccharomyces cerevisiae TBP. Specific Aim 1 is to investigate the function of RTF1, a protein that is important both for TATA site selection by TBP and for transcript elongation. RTF1 and associated proteins will be purified, RTF1-regulated genes will be identified using DNA microarrays, and genetic selections will be performed to identify extragenic suppressors of rtf1 mutations. In addition, the phosphorylation state of Pol II, which correlates with the transcription cycle, will be analyzed in rtf1 and other mutant strains. Specific Aim 2 is to elucidate the importance of TBP as a target for transcriptional regulatory factors in vivo. Chromatin immunoprecipitation and in vivo footprinting methods will be used to determine the effect of gene-specific and globally acting transcription factors on the recruitment of TBP to a highly regulated model promoter. Specific Aim 3 is to analyze two distinct classes of TBP mutants and gain insights into two fundamental aspects of Pol II transcription: orientation specific assembly of the preinitiation complex and regulation of transcription initiation. TBP mutants that exhibit reversed DNA binding polarity in vivo will be analyzed using in vitro DNA cleavage and transcription assays. TBP mutants that are altered in a particular subdomain of TBP and exhibit novel phenotypes, indicative of defects in transcriptional regulation, will be studied using both biochemical and genetic approaches. Since the proteins and mechanisms employed in the regulation of RNA polymerase II are conserved throughout eukaryotes, the information that is learned from these studies in yeast will significantly advance our understanding of transcription in humans, where a disruption in this process leads to important human diseases including cancer and AIDS.