Structure and Function of Gene Regulatory Complexes

描述（由申请人提供）：人体中的每个体细胞都包含相同的遗传信息，但通过对基因活性的精确控制，使分化细胞的不同功能成为可能。适当的基因调控对细胞的正常功能至关重要，人类疾病可以而且确实是由于正常基因调控的缺失或规避造成的。因此，我们的长期目标是了解真核细胞中基因表达是如何调控的。这项拨款申请的重点是转录共激活因子，它使tata结合蛋白（TBP）结合到TATA-box DMA结合位点，这是启动基因转录的关键步骤。重要数据表明，SAGA组蛋白乙酰转移酶（HAT）复合体通过将TBP传递到启动子和启动子区域的核小体乙酰化而发挥辅助激活因子的作用。尽管在过去十年中取得了重大进展，但这两种活性的分子机制尚不清楚。例如，我们并不确切地知道SAGA如何将TBP招募到核心启动子。同样，虽然我们确实了解HAT亚基如何识别组蛋白尾部底物，但我们对HAT或其他染色质酶如何识别其生理核小体底物却没有相应的了解。我们的具体目标就是要解决这些缺陷。我们特别建议：1。确定SAGA的Spt8亚基如何与TBP结合。我们将进行诱变和标签转移实验，以确定相互作用的TBP和Spt8表面。2. 确定SAGA如何将TBP招募到启动子TATA-box中。我们将开发和利用体外试验来研究SAGA如何将TBP传递给启动子的分子机制。3. 确定Ada2/Ada3/Gcn5和Piccolo/NuA4 HAT酶如何与核小体相互作用。我们将使用光亲和标记来识别靠近核小体的HAT复合物亚基，并通过缺失和突变研究来定义核小体结合和乙酰化过程中溴和色域的新功能。我们的生化研究使用确定的成分，通过解决其他方法不容易解决的机制问题，将补充大量可用的体内数据。

DESCRIPTION (provided by applicant): Each somatic cell in a human body contains the same genetic information, but the diverse function of differentiated cells is made possible through precise control of gene activity. Proper gene regulation is crucial for the normal function of a cell, and human diseases can and do result from loss or circumvention of normal gene regulation. Our long term objective is therefore to understand how gene expression is regulated in eukaryotic cells. This grant proposal focuses on transcriptional coactivators which enable the TATA-binding protein (TBP) to bind to its TATA-box DMA binding site, a critical step in initiating gene transcription. Significant data indicates that the SAGA histone acetyltransferase (HAT) complex functions as a coactivator by delivering TBP to the promoter and by acetylating nucleosomes in the promoter region. Despite major advances in the last decade, the molecular mechanism for either activity is not well-understood. For example, we do not know precisely how SAGA recruits TBP to the core promoter. Similarly, while we do understand how HAT subunits recognize histone tail substrates, we do not have an equivalent understanding of how HAT or other chromatin enzymes recognize their physiological nucleosome substrate. Our specific aims are directed to address these deficiencies. In particular, we propose to: 1. Determine how SAGA's Spt8 subunit binds to TBP. We will perform mutagenesis and label transfer experiments to define the TBP and Spt8 surfaces that interact. 2. Determine how SAGA recruits TBP to a promoter TATA-box. We will develop and utilize in vitro assays to examine the molecular mechanism for how SAGA delivers TBP to a promoter. 3. Determine how the Ada2/Ada3/Gcn5 and Piccolo/NuA4 HAT enzymes interact with nucleosomes. We will use photoaffinity labeling to identify HAT complex subunits in close proximity to nucleosomes, and deletion and mutagenesis studies to define novel functions for the bromo and chromodomains in the binding and acetylation of nucleosomes. Our biochemical studies using defined components will complement the considerable available in vivo data by addressing mechanistic questions not easily tackled by other approaches.