Molecular Analysis of a Yeast Transcriptional Regulator

描述（由申请人提供）： 项目摘要/摘要拟议项目的广泛的，长期的目标是阐明负责装配和活动的RNA聚合酶II（Pol II）preinitiation复合物（PIC）在启动子的机制。TATA结合蛋白（TBP）是PIC的核心成分，在体外能与DNA结合，并具有优异的稳定性。然而，基本上所有的TBP在酵母细胞中是高度动态的，在染色质上的停留时间只有几秒钟。来自体外观察的流行模型表明，一个稳定的启动子结合复合物促进转录重新启动，从而使激活状态永久化。相反，我们发现，动态营业额是至关重要的全球转录控制在体内。TBP的动态行为是Mot 1活性的结果，Mot 1是一种必需的、保守的、Snf 2/Swi 2相关的ATP酶，其使用ATP水解的能量将TBP从DNA中解离。该提案的目标是使用Mot 1作为模型，以更好地了解这类关键酶的成员如何使用ATP水解来破坏蛋白质-DNA相互作用，并了解为什么Mot 1介导的动力学对体内转录至关重要。在第一个目标中，Mot 1生化系统和最近的结构数据将被利用来测试Mot 1催化机制的特定模型。新出现的证据表明，细胞与细胞的基因表达变异（“噪音”）影响了广泛的细胞反应的压力和发展的线索，但机制调节噪音还没有得到很好的理解。初步结果表明，Mot 1在抑制整个细胞群的转录噪声中具有以前未被认识到的作用。在目标2中，我们将定义Mot 1和其他相关的全球监管机构在建立噪声水平方面的范围和功能，并将这种变化与PIC动态相关联。在目标3中，我们将定义Mot 1和FACT之间新发现的功能关系，FACT是染色质结构和动力学的重要调节因子。除了确定Mot 1和FACT如何在体内一起工作外，Mot 1在染色质结构调节中的新功能也将得到解决，包括Mot 1与核小体结合和调节FACT与核小体相互作用的意外能力。 精确的转录控制对于正常细胞生长和发育是必不可少的，并且许多转录缺陷与包括癌症在内的人类疾病有关。人Snf 2/Swi 2相关蛋白质复合物的缺陷已知是某些癌症、科凯恩综合征、α-地中海贫血和最常见形式的X-连锁精神发育迟滞的促成因素。

DESCRIPTION (provided by applicant): Project Summary/Abstract The broad, long-term objectives of the proposed project are to elucidate mechanisms responsible for assembly and activity of RNA polymerase II (Pol II) preinitiation complexes (PICs) at promoters. The TATA- binding protein (TBP), a central component of the PIC, can bind to DNA in vitro with exceptional stability. However, essentially all of the TBP in yeast cells is highly dynamic, with residence times on chromatin of just a few seconds. Prevailing models derived from in vitro observations posit that a stable promoter-bound complex facilitates transcription reinitiation and thereby perpetuates the activated state. In contrast, we find that dynamic turnover is critical for global transcriptional control in vivo. TBP's dynamic behavior is a consequence of the activity of Mot1, an essential, conserved, Snf2/Swi2-related ATPase that uses the energy of ATP hydrolysis to dissociate TBP from DNA. The goals of this proposal are to use Mot1 as a model to better understand how members of this critical class of enzymes use ATP hydrolysis to disrupt protein-DNA interactions, and to understand why Mot1-mediated dynamics are essential for transcription in vivo. In the first Aim, the Mot1 biochemical system and recent structural data will be exploited to test a specific model for the Mot1 catalytic mechanism. Emerging evidence indicates that cell-to-cell gene expression variation ("noise") impacts a wide range of cellular responses to stress and developmental cues, but mechanisms regulating noise are not well understood. Preliminary results show that Mot1 has a previously unappreciated role in suppressing transcriptional noise across a cell population. In Aim 2, we will define the scope and function of Mot1 and other related global regulators in establishing noise levels, and correlate this variation with PIC dynamics. In Aim 3, we will define a newly discovered functional relationship between Mot1 and FACT, an essential regulator of chromatin structure and dynamics. In addition to determining how Mot1 and FACT work together in vivo, new functions of Mot1 in chromatin structure regulation will be addressed, including the unexpected ability of Mot1 to bind to nucleosomes and to regulate the interaction of FACT with nucleosomes. Precise transcriptional control is essential for normal cell growth and development, and numerous transcriptional defects have been linked to human diseases including cancer. Defects in human Snf2/Swi2- related protein complexes are known contributors to certain cancers, Cockayne's Syndrome, alpha- thalassemia, and the most common form of X-linked mental retardation.