Molecular Analysis of a Yeast Transcriptional Regulator

描述(申请人提供)：拟议项目的长期目标是阐明Mot1的作用机制和在体内的功能，Mot1是酿酒酵母中一种基本的、保守的转录调节因子。Mot1与TATA结合蛋白(TBP)形成独特的复合体，在全基因组范围内调节TBP的功能和DMA结合活性。Mot1是一个进化保守的核ATPase大家族(Snf2/Swi2家族)的成员，几乎参与了DMA代谢的各个方面。人类Snf2/Swi2相关蛋白复合体的缺陷会导致某些癌症、Cockayne综合征、α-地中海贫血以及最常见的X连锁智力低下。尽管蛋白质在这个家族中普遍存在，但它们的分子作用机制尚不清楚，也不清楚其中许多蛋白质在体内扮演什么角色。Mot1的S ATPase活性是激活或抑制体内特定基因转录所必需的。Mot1可以在依赖于ATP的反应中解离TATA结合蛋白(TBP)-DNA复合体，这与其作为抑制物的作用是一致的。我们认为Mot1也可以通过这种活性来取代启动子上稳定结合的转录不活跃的TBP形式来激活转录。这种TBP回收是为了为转录复合体组装提供质量控制，并确保有足够的自由TBP池用于全基因组范围内启动子活性的动态控制。Mot1介导的激活也涉及到与NC2和SAGA复合体之间鲜为人知的合作。将使用生化、分子生物学和遗传学方法来测试循环模型，并定义Mot1、NC2和SAGA如何在转录控制中合作。生物化学方法将被用来测试Motl催化机制的一个特定的和普遍适用的模型，在该模型中，ATP水解驱动两种不同构象之间的ATPase的相互转化。对Mot1功能的分析将有助于更好地理解转录复合体的动力学，并将为Snf2/Swi2相关蛋白质如何将ATP水解与机械力的产生相结合提供一般的见解。

DESCRIPTION (provided by applicant): The long-term objectives of the proposed project are to elucidate the mechanism of action and in vivo function of Mot1, an essential, conserved, transcriptional regulator in the yeast Saccharomyces cerevisiae. Mot1 forms a unique complex with the TATA-binding protein (TBP) and regulates TBP's function and DMA binding activity on a genome-wide scale. Mot1 is a member of a large family of evolutionary conserved nuclear ATPases (the Snf2/Swi2 family) involved in virtually all aspects of DMA metabolism. Defects in human Snf2/Swi2-related protein complexes contribute to certain cancers, Cockayne's Syndrome, a-thalassemia, and the most common form of X-linked mental retardation. Despite the ubiquitous occurrence of proteins in this family, their molecular mechanisms of action are not understood in detail, nor is it understood what roles many of these proteins play in vivo. Mot1 's ATPase activity is required to activate or repress transcription of specific genes in vivo. Consistent with its role as a repressor, Mot1 can dissociate TATA-binding protein (TBP)-DNA complexes in an ATP-dependent reaction. We propose that Mot1 can also activate transcription by using this activity to displace stably-bound, transcriptionally inactive forms of TBP from promoters. Such TBP recycling is proposed to provide quality control for transcription complex assembly and to ensure an adequate pool of free TBP for dynamic control of promoter activity genome-wide. Mot1- mediated activation also involves poorly understood cooperation with the NC2 and SAGA complexes. Biochemical, molecular biological, and genetic approaches will be used to test the recycling model and to define how Mot1, NC2 and SAGA cooperate in transcriptional control. Biochemical approaches will be used to test a specific and generally applicable model for Motl's catalytic mechanism in which ATP hydrolysis drives the interconversion of the ATPase between two different conformational forms. The proposed analysis of Mot1 function will lead to a better understanding of transcription complex dynamics and will provide general insight into how Snf2/Swi2-related proteins couple ATP hydrolysis to the generation of mechanical force.