TRANSCRIPTION FACTOR MUTANTS OF YEAST

酵母转录因子突变体

• 批准号: 6372713
• 负责人: KAREN M ARNDT
• 金额: $ 10.06万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6372713
• 关键词: DNA directed RNA polymerase; Saccharomyces cerevisiae; binding proteins; chromatin; fungal genetics; genetic promoter element; immunoprecipitation; microarray technology; mutant; phosphorylation; protein structure function; transcription factor

My long-term research objectives are to identify factors that play important roles in transcription by RNA polymerase II (Pol II) in vivo and to determine the mechanisms by which these factors act. The proposed studies reflect my longstanding interests in gene regulation and commitment to biomedical research. The experiments will be performed in a highly interactive and supportive research environment, and my progress will be greatly aided by institutional commitments to alleviate teaching and committee responsibilities. My immediate research goals center on transcription initiation and early post- initiation events. The Specific Aims represent extensions of our previous studies on Saccharomyces cerevisiae TATA box-binding protein (TBP). TBP binds directly to the TATA box at Pol II promoters and initiates a cascade of events that culminate in 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. Specific Aim 1 is to investigate the function of Rtf1, a novel protein 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 suppressors of rtf1 mutations. In addition, the phosphorylation state of Pol II, which correlates with the transcription cycle, will be analyzed in various mutant strains. Specific Aim 2 is to elucidate the importance of TBP as a target for 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 transcription complex and regulation of initiation. TBP mutants that exhibit reversed DNA binding polarity in vivo will be analyzed using DNA cleavage and transcription assays. TBP mutants that are altered in a particular subdomain of TBP and exhibit phenotypes indicative of transcriptional defects will be studied. The results from this work will provide a solid foundation for the continued dissection of transcription initiation and elongation in future years. Since the proteins and mechanisms employed in the regulation of Pol II are highly conserved, the information that is learned from these studies in yeast will significantly advance our understanding of transcription in humans, where an alteration in this process leads to important human diseases including AIDS.

我的长期研究目标是确定在体内RNA聚合酶II（Pol II）转录中发挥重要作用的因子，并确定这些因子的作用机制。 这些研究计划反映了我对基因调控的长期兴趣和对生物医学研究的承诺。 实验将在一个高度互动和支持性的研究环境中进行，我的进步将大大有助于机构的承诺，以减轻教学和委员会的责任。 我的近期研究目标集中在转录起始和起始后的早期事件上. 具体目的是我们以前对酿酒酵母TATA盒结合蛋白（TBP）的研究的延伸。 TBP在Pol II启动子处直接与TATA盒结合，并启动一系列事件，最终导致RNA信息。 因此，了解控制TBP的因素是了解真核生物基因表达调控的关键一步。 具体目标1是研究Rtf1的功能，Rtf1是一种新的蛋白质，对于TATA位点选择和转录延长都很重要。将纯化Rtf1和相关蛋白，使用DNA微阵列鉴定Rtf1调节基因，并进行遗传选择以鉴定rtf1突变的抑制因子。 此外，将在各种突变株中分析与转录周期相关的Pol II的磷酸化状态。 具体目标2是阐明TBP作为体内调节因子靶点的重要性。 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的转录因子对TBP募集到高度调节的模型启动子的影响。具体目标3是分析两种不同类型的TBP突变体，并深入了解Pol II转录的两个基本方面：转录复合物的定向特异性组装和起始调控。 将使用DNA切割和转录测定来分析在体内表现出逆转的DNA结合极性的TBP突变体。 将研究在TBP的特定亚结构域中改变并表现出指示转录缺陷的表型的TBP突变体。 这些结果将为今后进一步研究转录起始和延伸提供坚实的基础。 由于在Pol II的调节中所采用的蛋白质和机制是高度保守的，因此从这些酵母研究中所学到的信息将大大促进我们对人类转录的理解，其中该过程的改变导致重要的人类疾病，包括艾滋病。