Molecular Mechanism of Gene Transcription Activation

 描述（由申请人提供）：我们试图确定RNA聚合酶II控制真核mRNA基因转录的分子机制。mRNA生物合成的缺陷可能导致人类疾病，如癌症、糖尿病和肥胖症。因此，必须解剖基因表达的第一步，mRNA基因转录，以便我们能够理解正常和病理状态。 我们正在进行的工作利用遗传上易于处理的面包酵母，酿酒酵母，作为一个模型来阐明的多重作用，高度进化保守的转录因子复合物TFIID在mRNA基因转录中发挥作用。TFIID由15个亚基组成（TATA盒结合蛋白，TBP，加上14个TBP相关因子，Taf 1 <$Taf14）。TFIID在六种所谓的通用转录因子（GTF：TFIIA、B、D、E、F、H）中是独特的，因为它不仅是启动子指导的前起始复合物（PIC）形成所需的（实际上TFIID是识别并结合TATA启动子元件的GTF），而且TFIID还在某些后生动物mRNA编码基因上充当转录共激活因子。TFIID存在于从酵母到人类的超过90%的mRNA编码基因的转录中，并且其功能是转录所需的。 我们最近的工作表明，酵母TFIID作为转录因子阻遏激活蛋白1（Rap 1）通过与TFIIA合作的转录共激活因子。Rap 1是激活100多个编码组成核糖体的蛋白质补体的基因转录的必需转因子;核糖体水平和翻译普遍限制细胞增殖的速率。Rap 1，RNA聚合酶II和六个GTF的突变都是致命的，结果表明所有这些蛋白质都有助于关键的细胞功能。 我们建议采用多方面的方法结合生物化学，遗传学，蛋白质组学和结构的方法来阐明如何相互作用的TFIID和Rap 1与彼此，增强子-启动子DNA，并与GTF和RNA聚合酶II，导致基因转录的精确和受控的激活实验。最终，我们计划在一个自然的， 体内组装的mRNA编码基因。我们相信，我们的工作将提供新的和全球性的见解，了解mRNA基因转录调控机制。

 DESCRIPTION (provided by applicant): We seek to define the molecular mechanisms controlling eukaryotic mRNA gene transcription by RNA Polymerase II. Defects in mRNA biosynthesis may lead to human maladies such as cancer, diabetes and obesity. Thus, it is imperative to dissect the very first step in gene expression, mRNA gene transcription, so that we can understand both normal and pathological states. Our ongoing work utilizes the genetically-tractable baker's yeast, Saccharomyces cerevisiae, as a model to elucidate the multiple roles that the highly evolutionarily-conserved transcription factor complex TFIID plays in mRNA gene transcription. TFIID is composed of 15 subunits (the TATA box Binding Protein, TBP, plus 14 TBP-associated factors, Taf1¿Taf14). TFIID is unique among the six so-called General Transcription Factors (GTFs: TFIIA, B, D, E, F, H) in that not only is it required for promoter-directed Pre-Initiation Complex (PIC) formation (indeed TFIID is the GTF that recognizes and binds the TATA promoter element), but TFIID also acts as a transcriptional coactivator on certain metazoan mRNA-encoding genes. TFIID is resident on, and its function is required for, the transcription of over 90% of mRNA encoding genes from yeast to humans. Our recent work has shown that yeast TFIID serves as a transcriptional coactivator for the transcription factor Repressor activator protein 1 (Rap1) via collaboration with TFIIA. Rap1 is an essential transfactor that activates transcription of the 100+ genes that encode the complement of proteins composing the ribosome; ribo- some levels and translation are universally rate-limiting for cellular proliferation. Null mutations of Rap1, RNA polymerase II and the six GTFs are all lethal, results indicating that all of these proteins contribute key cellular functions. We propos experiments that employ a multifaceted approach combining biochemical, genetic, proteomic, and structural methods to elucidate how the interplay of TFIID and Rap1 with each other, with enhancer- promoter DNA, and with GTFs and RNA Polymerase II, leads to the precise and controlled activation of gene transcription. Ultimately we plan to study this process on a natural, in vivo-assembled, mRNA-encoding gene. We believe our work will provide novel and global insights to understand mRNA gene transcription regulatory mechanisms.