Structure and Function of Transcription Complexes

描述（由申请人提供）：开发新型有效的癌症治疗方法需要对调节蛋白如何开启和关闭介导人类细胞中细胞生长、瘤形成、转移、血管生成等的基因表达模式有基本的了解。 全面了解多亚基转录复合物的功能和结构，负责指导控制人类基因表达的调控信号的巨大网络构成了一个巨大的挑战，形成了这项资助提案的基础。 为了解决研究多亚基转录复合物所固有的困难的结构/功能问题，我们建议开展协调和跨学科的努力，以确定关键的多亚基转录复合物的3D结构，并解开调节人类和动物细胞中基因转录的机制。 该P01计划项目资助的一个关键方面是建立跨学科和研究机构的高度互动合作，以研究大型多亚基转录复合物的结构/功能关系。 通过结合几位研究者的专业知识，我们建议实施一系列互补的生物化学和生物物理方法，包括：项目1，激活剂/辅激活剂复合物的电子显微镜和单颗粒重建以及染色质重塑活动;项目2，蛋白质生物化学：蛋白质相互作用和大型多亚基转录复合物的X射线晶体学（TFIIA和TFIID）;和项目3，转录组分与DNA和启动子元件的动态组装的单分子FRET分析。 该提案的一个主要组成部分将是建立一个生物化学和核心蛋白质实验室，作为神经中枢和信息交换所，用于生成三个研究项目中的每个项目所使用的许多细胞系、表达克隆、蛋白质复合物和纯化策略。 因此，我们的目标是开发一种高度协同和协调一致的努力，通过使用相同的分子试剂，但应用不同的和互补的研究策略来解剖负责基因控制导致癌症的核心机制，来进行一系列开创性的相互依赖的实验。

DESCRIPTION (provided by applicant): The development of novel and effective treatments for cancer requires a fundamental understanding of how regulatory proteins switch on and off gene expression patterns that mediate cell growth, neoplasia, metastasis, angiogenesis, etc. in human cells. A comprehensive understanding of the function and structure of multi-subunit transcription complexes responsible for directing the vast networks of regulatory signals controlling gene expression in humans poses a formidable challenge that forms the basis of this grant proposal. To tackle the difficult structure/function problems inherent with studying multi-subunit transcription complexes, we propose to launch a coordinated and interdisciplinary effort to determine the 3D structures of key multi-subunit transcription complexes and unravel the mechanisms that regulate gene transcription in human and animal cells. A key aspect of this P01 Program Project Grant will be to establish highly interactive collaborations across disciplines and research institutes to study the structure/function relationships of large multi-subunit transcription complexes. By combining the expertise of several investigators, we propose to implement a battery of complementary biochemical and biophysical methods including: Project 1, electron microscopy and single particle reconstruction of activator/co-activator complexes as well as chromatin remodeling activities; Project 2, biochemistry of protein:protein interactions and X-ray crystallography of large multi-subunit transcription complexes (TFIIA and TFIID); and Project 3, single molecule FRET analysis of the dynamic assembly of transcription components with DNA and promoter elements. A major component of this proposal will be the establishment of a biochemistry and core protein laboratory to serve as the nerve center and clearinghouse for generating the many cell lines, expression clones, protein complexes, and purification strategies to be used by each of the three Research Projects. Thus, our goal is to develop a highly synergistic and concerted effort to carry out a pioneering set of interdependent experiments by using the same molecular reagents but applying distinct and complementary research strategies to dissect the core machinery responsible for gene control leading to cancer.