Molecular Analysis of a Yeast Transcriptional Regulator

描述（由申请人提供）：拟议项目的长期目标是阐明Mot 1的作用机制和体内功能，Mot 1是酿酒酵母中一种重要的保守转录调节因子。Mot 1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。Mot 1是一个进化保守的核ATP酶大家族（Snf 2/Swi 2家族）的成员，参与DMA代谢的几乎所有方面。人类Snf 2/Swi 2相关蛋白质复合物的缺陷导致某些癌症、科凯恩综合征、α-地中海贫血和最常见的X连锁精神发育迟滞形式。尽管在这个家族中蛋白质普遍存在，但它们的分子作用机制尚未详细了解，也不了解这些蛋白质中的许多在体内发挥什么作用。Mot 1的ATP酶活性是激活或抑制体内特定基因转录所必需的。与其作为阻遏物的作用一致，Mot 1可以在ATP依赖性反应中解离TATA结合蛋白（TBP）-DNA复合物。我们建议Mot 1也可以通过使用这种活性来激活转录，以取代稳定结合的，转录失活形式的TBP启动子。提出这种TBP再循环为转录复合物组装提供质量控制，并确保足够的游离TBP库用于全基因组启动子活性的动态控制。Mot 1介导的激活还涉及与NC 2和佐贺复合物的合作。生物化学，分子生物学和遗传学的方法将被用来测试回收模型，并确定如何Mot 1，NC 2和佐贺在转录控制合作。生物化学的方法将被用来测试一个特定的和普遍适用的模型Motl的催化机制，其中ATP水解驱动两种不同的构象形式之间的ATP酶的相互转换。Mot 1功能的拟议分析将导致更好地理解转录复合物的动力学，并将提供一般的洞察Snf 2/Swi 2相关蛋白如何耦合ATP水解的机械力的产生。

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.