Structure and Function of Transcription Complexes

描述(申请人提供)：开发新的和有效的癌症治疗方法需要对调节蛋白如何开启和关闭基因表达模式的基本了解，这些基因表达模式调节细胞生长、肿瘤、转移、血管生成等。全面了解多亚基转录复合体的功能和结构，负责指导控制人类基因表达的巨大调控信号网络，构成了这项拨款提案的基础，构成了一个艰巨的挑战。为了解决研究多亚单位转录复合体所固有的结构/功能问题，我们建议开展一项协调和跨学科的努力，以确定关键的多亚单位转录复合体的三维结构，并揭示人类和动物细胞中基因转录的调控机制。P01计划项目拨款的一个关键方面将是建立跨学科和研究机构的高度互动的合作，以研究大型多亚基转录复合体的结构/功能关系。通过结合几位研究人员的专业知识，我们提议实施一系列互补的生化和生物物理方法，包括：项目1，电子显微镜和单粒子重建激活剂/共激活剂复合体以及染色质重塑活动；项目2，蛋白质的生物化学：蛋白质相互作用和大型多亚单位转录复合体的X射线结晶学(TFIIA和TFIID)；以及项目3，转录成分与DNA和启动子元件动态组装的单分子FRET分析。这项提议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中心，产生三个研究项目中每一个都将使用的许多细胞系、表达克隆、蛋白质复合体和纯化策略。因此，我们的目标是开发一种高度协同和协调的努力，通过使用相同的分子试剂，但应用不同和互补的研究策略来剖析导致癌症的基因控制的核心机制，从而开展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.