Molecular Analysis of a Yeast Transcriptional Regulator

项目描述（由申请人提供）：该项目的长期目标是阐明酵母中重要的、保守的转录调节因子Mot1的作用机制和体内功能。Mot1与tata结合蛋白（TBP）形成独特的复合物，并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atp酶大家族（Snf2/Swi2家族）的成员，几乎参与了DMA代谢的所有方面。人类Snf2/ swi2相关蛋白复合物的缺陷会导致某些癌症、柯凯因综合征、地中海贫血和最常见的x连锁智力迟钝。尽管这个家族的蛋白质无处不在，但它们的分子作用机制尚不清楚，也不清楚这些蛋白质在体内发挥的作用。Mot1的atp酶活性是激活或抑制体内特定基因转录所必需的。与其作为抑制因子的作用一致，Mot1可以在atp依赖的反应中解离tata结合蛋白(TBP)-DNA复合物。我们提出，Mot1也可以利用这种活性来取代启动子中稳定结合的、转录不活跃的TBP形式，从而激活转录。提出这种TBP回收是为了提供转录复合物组装的质量控制，并确保有足够的自由TBP池用于全基因组启动子活性的动态控制。Mot1介导的激活也涉及到与NC2和SAGA复合物的合作。将使用生化、分子生物学和遗传学方法来测试循环模型，并确定Mot1、NC2和SAGA如何在转录控制中合作。生化方法将被用来测试一个特定的和普遍适用的模型，用于Motl的催化机制，其中ATP水解驱动ATP酶在两种不同构象形式之间的相互转化。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，并将提供对Snf2/ swi2相关蛋白如何将ATP水解偶联以产生机械力的一般见解。

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.