TRANSCRIPTION FACTOR MUTANTS OF YEAST

描述（改编自研究者摘要）：长期 拟议研究的目标是确定在这方面起重要作用的因素， RNA聚合酶II在体内转录中的作用，并确定 这些因素控制这一过程的机制。的中心焦点 这种补助金是TATA盒结合蛋白（TBP），它在 在所有三种核RNA聚合酶的转录起始中。在促销员 由Pol II转录，TBP与TATA盒的结合是转录的第一步。 这是一系列事件的级联反应，最终导致RNA信息的产生。 因此，了解控制TBP的因素是关键的一步 了解真核生物基因表达的调控。的 特定目的代表了对酵母菌先前研究的扩展 酿酒TBP。具体目的1是研究RTF1的功能， 该蛋白对于TBP的TATA位点选择和 转录延长将纯化RTF 1和相关蛋白， RTF1调控的基因将使用DNA微阵列进行鉴定， 将进行选择以鉴定rtf1的基因外抑制因子 突变。此外，与细胞凋亡相关的Pol II的磷酸化状态 与转录周期，将分析在rtf1和其他突变 菌株具体目标2是阐明TBP作为靶点的重要性， 体内转录调节因子。染色质免疫沉淀和 体内足迹法将用于确定 基因特异性和全局作用转录因子对募集 TBP是一个高度调节的模型启动子。具体目标3是分析两个 不同类别的TBP突变体，并深入了解两个基本方面 Pol II转录的方向特异性组装 复合物和转录起始的调节。TBP突变体， 将使用体外DNA分析体内逆转的DNA结合极性 切割和转录测定。TBP突变体在一个特定的 TBP亚结构域，并表现出新的表型，表明TBP 转录调控，将使用生物化学和遗传学方法进行研究。 接近。 由于蛋白质和机制的调控， RNA聚合酶II在整个真核生物中是保守的， 从这些酵母研究中学到的知识将大大促进我们的理解 在人类的转录过程中，这一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.