STRUCTURE AND FUNCTION OF HUMAN TRANSLATION COMPLEXES

真核翻译起始因子控制mRNA与40 S核糖体装配的几个步骤 在蛋白质合成的开始。其中两个因子eIF 2和eIF 3几乎普遍存在于 这是帽依赖性和帽非依赖性起始所需的。真核起始因子eIF 2是异源三聚体 负责将起始tRNA带到核糖体-mRNA组装体，并代表关键靶点之一 由激酶负向翻译调节。起始因子eIF 3包括至少12个不相同的 它是m7 G-cap-和IRES-驱动的mRNA翻译所必需的。内部核糖体 丙型肝炎病毒（HCV）的进入位点（IRES）在翻译起始期间利用eIF 2和eIF 3两者， IRES和eIF 3之间的直接接触对于有效的病毒翻译是必需的。这项建议旨在 了解这些因素活动的生化和结构基础。我们将开发方法 用于大规模纯化这些因子，并将分析其单独和功能内的组成 核糖体复合物由HCV IRES使用定量质谱法定义。康贝特人将以 通过分离和分析翻译复合物， 在体外和体内结合IRES的野生型和突变形式。最后，我们计划确定分子 eIF 2和eIF 3的结构，提供了一个结构 解释IRES介导的和帽依赖的翻译过程中发生的相互作用的框架。 与Gate、Harris、Hershey和萨尔诺实验室以及 拟议的质谱核心实验室将由Leary教授领导，是这些实验室的一个组成部分。 实验

Eukaryotic translation initiation factors control several steps in the assembly of mRNA with 40S ribosomal subunits at the start of protein synthesis. Two of these factors, elF2 and elF3, are almost universally required for cap-dependent and cap-independent initiation. Eukaryotic initiation factor elF2 is a heterotrimer responsible for bringing initiator tRNA to ribosome-mRNA assemblies, and represents one of the key targets of negative translation regulation by kinases. Initiation factor elF3 includes at least twelve non-identical subunits and is required for translation of most m7G-cap- and IRES-driven mRNAs. The internal ribosome entry site (IRES) of hepatitis C virus (HCV) utilizes both elF2 and elF3 during translation initiation, and a direct contact between the IRES and elF3 is essential for efficient viral translation. This proposal aims to understand the biochemical and structural basis for the activities of these factors. We will develop methods for large-scale purification of these factors and will analyze their composition both alone and within functional ribosomal complexes defined by the HCV IRES using quantitative mass spectrometry. We will determine the role of elF3 in HCV IRES-mediated translation initiation by isolating and analyzing translation complexes that bind the wild type and mutant forms of the IRES in vitro and in vivo. Finally, we plan to determine molecular structures of elF2 and elF3 using both X-ray crystallography and electron microscopy, providing a structural framework for interpreting interactions that occur during both IRES-mediated and cap-dependent translation. Direct collaboration and interactions with the Gate, Harris, Hershey and Sarnow laboratories, as well as the proposed Mass Spectrometry Core Laboratory to be directed by Prof. Leary, is an integral part of these experiments.