Molecular Genetic Analysis of Yeast TAFs

描述（由申请人提供）：我们研究的目的是了解RNA聚合酶II（Pol II）控制真核mRNA基因转录的分子机制。如果我们要真正了解正常和病理过程和条件，并最终干预治疗疾病，对转录调控机制的全面了解是绝对必要的。我们的工作利用遗传上易处理的酿酒酵母（Saccharomyces cerevisiae）或面包酵母（Baker's Yeast）作为模型，用于阐明通用转录因子（GTF）TFIID在由Pol II进行的mRNA基因转录的复杂过程中所起的详细作用。我们计划的实验代表了我们对TBP的长期研究的结果，TBP是TATA盒结合蛋白，一种关键的蛋白质因子，它与mRNA编码基因的启动子结合，以启动最终在Pol II转录和基因表达中的事件链。总细胞TBP的显著部分仅在与称为TBP相关因子（TAF）的额外TBP结合蛋白的RNA聚合酶特异性集合相关联时才起作用。我们已经表明，在酵母细胞中有14个完整的Pol II TAF或TAFII，与TBP结合形成TFIID多亚基复合物。我们和其他人，与酵母和哺乳动物细胞的工作，已经表明，TFIID复合物密切参与调控事件，控制某些mRNA编码基因的转录。因此，了解TBP-TAF复合物的组成和功能将是阐明基因转录如何在分子水平上控制的关键。我们计划用于分析TFIID的方法将是多方面的，并将联合收割机生物化学，生物物理和遗传技术，以检查这些因素与TBP，彼此，额外的GTF，反式激活蛋白，启动子DNA和RNA聚合酶II的相互作用。这些实验的成功完成将极大地增加我们对mRNA基因转录和疾病的所有重要过程的理解。

DESCRIPTION (provided by applicant): The goal of our research is to understand the molecular mechanisms controlling eukaryotic mRNA gene transcription by RNA Polymerase II (Pol II). A comprehensive understanding of transcriptional regulatory mechanisms is absolutely essential if we are to truly appreciate both normal and pathological processes and conditions and ultimately intervene to cure disease. Our work utilizes the genetically tractable Saccharomyces cerevisiae, or Baker's Yeast, as a model for elucidating the detailed role that the general transcription factor (GTF) TFIID plays in the complicated process of mRNA gene transcription by Pol II. Our planned experiments represent the outgrowth of our long-term study of TBP, the TATA-box Binding Protein, a critical protein factor that binds to the promoter of mRNA-encoding genes in order to set into motion the chain of events culminating in Pol II transcription and thus gene expression. A significant fraction of the total cellular TBP is only functional when associated with RNA Polymerase-specific collections of additional TBP binding proteins known as TBP Associated Factors (TAFs). We have shown that in yeast cells there are fourteen integral Pol II TAFs, or TAFIIs, that associate with TBP to form the TFIID multi-subunit complex. We and others, working both with yeast and mammalian cells, have shown that the TFIID complex is intimately involved in the regulatory events that control the transcription of certain mRNA encoding genes. Therefore, understanding the composition and function of TBP-TAF complexes will be key to elucidating how gene transcription is controlled at the molecular level. The approaches that we plan to use to analyze TFIID will be multi-faceted and will combine biochemical, biophysical and genetic techniques to examine the interplay of these factors with TBP, each other, additional GTFs, trans-activator proteins, promoter DNA, and RNA polymerase II. Successful completion of these experiments will greatly increase our understanding of the all-important process of mRNA gene transcription and hence disease.