Program in Virus Translational Control

描述（由申请人提供）：开发新的有效的病毒感染治疗方法需要对病毒如何在感染的早期阶段招募宿主细胞翻译机制进行病毒蛋白合成有一个基本的了解。了解大型多亚基翻译起始因子和病毒rna如何控制核糖体的结合和活性是一项艰巨的挑战，这是本资助提案的基础。为了解决翻译研究中固有的机制和结构问题，我们建议开展协调和跨学科的工作，以确定关键起始因子复合物的组成、分子间相互作用和结构，并揭示调节人类细胞和病毒中蛋白质合成的机制。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之间的高度互动合作，以研究控制翻译起始的关键复合物的化学、结构和机制特性。通过结合几位研究人员的专业知识，我们建议实施一系列互补的生物物理和生化方法，包括：Doudna和Cate的项目，翻译起始因子和复合物的冷冻电子显微镜和x射线晶体学；好时（Hershey）项目，利用病毒和细胞翻译成分进行体内和体外翻译试验；和Project by Sarnow，病毒翻译复合物的纯化和分析。该提案的一个主要组成部分将是建立一个大型多亚基复合物质谱分析的核心实验室，该实验室将作为分析翻译起始过程中涉及的许多蛋白质和蛋白质- rna复合物的神经中枢和交换中心。该核心实验室将被所有五个研究项目广泛使用，以确定分别构成和控制人类细胞和病毒翻译活性的大分子和翻译后修饰。因此，我们的目标是开发一个高度协同和协调的努力，通过使用重叠的分子试剂，但应用独特和互补的研究策略来解剖正常和病毒感染的哺乳动物细胞中负责蛋白质合成的核心机制，开展一套开创性的相互依赖的实验。

DESCRIPTION (provided by applicant): The development of novel and effective treatments for viral infections requires a fundamental understanding of how viruses recruit the host cell translational machinery for viral protein synthesis during the early stages of infection. Understanding how large multi-subunit translation initiation factors and viral RNAs control the binding and activity of ribosomes poses a formidable challenge that forms the basis of this grant proposal. To tackle the difficult mechanistic and structural problems inherent in studying translation, we propose to launch a coordinated and interdisciplinary effort to determine the compositions, intermolecular interactions and structures of key initiation factor complexes and unravel the mechanisms that regulate protein synthesis in human cells and viruses. A key aspect of this P01 Program Project Grant will be to establish highly interactive collaborations across disciplines and research institutions to study the chemical, structural and mechanistic properties of key complexes controlling translation initiation. By combining the expertise of several investigators we propose to implement a battery of complementary biophysical and biochemical approaches including: Projects by Doudna and Cate, cryo-electron microscopy and X-ray crystallography of translation initiation factors and complexes; Project by Hershey, in vitro and in vivo translation assays with viral and cellular translation components; and Project by Sarnow, purification and analysis of viral translation complexes. A major component of this proposal will be the establishment of a core laboratory for mass spectrometry of large multi-subunit complexes that will serve as the nerve center and clearinghouse for analyzing the many protein and protein-RNA complexes involved in translation initiation. This core laboratory will be used extensively by all five Research Projects to identify the macromolecules and post-translational modifications that, respectively, comprise and control translation activity in human cells and viruses. Thus, our goal is to develop a highly synergistic and concerted effort to carry out a pioneering set of interdependent experiments by using overlapping molecular reagents but applying distinct and complementary research strategies to dissect the core machinery responsible for protein synthesis in normal and virally-infected mammalian cells.