EIF3 STRUCTURE /FUNCTION IN INITIATION AT IRESS

IRESS 启动时的 EIF3 结构/功能

• 批准号: 7299444
• 负责人: JOHN W HERSHEY
• 金额: $ 12.13万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7299444
• 关键词: binding sites; cell growth regulation; cell line; cell proliferation; dengue virus; genetic translation; globin; hepatitis C virus; human genetic material tag; intermolecular interaction; mass spectrometry; messenger RNA; phosphorylation; posttranslational modifications; protein biosynthesis; protein structure function; ribosomes; tissue /cell culture; transfection; translation factor; virus RNA; virus genetics; virus infection mechanism

The major goal of the project is to understand how the human translation initiation factor elF3 contributes to the initiation mechanism and to the regulation of viral mRNAs, especially Hepatitis C Virus (HCV) mRNA that initiates translation by ribosome binding to an internal entry site (IRES). The structure of elF3, a factor comprising 12 non-identical subunits, will be probed by isolating a variety of sub-complexes formed in baculovirus-infected insect cells. The binding of elF3 and its sub-complexes to the HCV IRES will be defined quantitatively. A highly active partially fractionated mammalian cell lysate system dependent on purified elF3 will be constructed with (5-globin mRNA. This system will be used to define the role of elF3 and its subcomplexes in the translation of HCV mRNA. An in vitro translation system also will be constructed that utilizes all of the purified initiation factors, in order to define which factors are required for the translation of HCV and Dengue virus mRNAs. These studies will determine to what extent the viral mRNAs differ from capped/polyadenylated mRNAs in their initiation mechanisms, and may identify targets for therapeutic intervention of infections by these viruses. Considerable evidence indicates that elF3 plays an important role in regulating protein synthesis and cell proliferation. elF3, like other initiation factors, is phosphorylated on numerous subunit proteins. The sites of phosphorylation will be identified by mass spectroscopy, and the effects of phosphorylation on function will be tested in the translation assays. Mutant forms will be constructed and tested, with phosphorylation sites substituted with alanine to prevent phosphorylation, and with glutamate or aspartate to mimic phosphorylation. The experiments are designed to elucidate the mechanism of action of elF3 and the role of phosphorylation in regulating its activity during initiation of capped and IRES-driven mRNAs. The results may explain how disfunction or disregulation of elF3 may affect the control of cell proliferation, resulting in celt malignancy.

该项目的主要目标是了解人类翻译起始因子eIF 3如何发挥作用， 启动机制和调节病毒mRNA，特别是丙型肝炎病毒（HCV）mRNA 其通过核糖体结合到内部进入位点（IRES）来启动翻译。eIF 3的结构，一个因子 包括12个不相同的亚基，将通过分离形成的各种亚复合物来探测， 杆状病毒感染的昆虫细胞。将定义eIF 3及其亚复合物与HCV IRES的结合 数量上。依赖于纯化eIF 3的高活性部分分级哺乳动物细胞裂解物系统 将用β-珠蛋白mRNA构建。该系统将用于定义eIF 3及其亚复合物的作用 HCV mRNA的翻译。还将构建体外翻译系统， 利用所有纯化的起始因子，以确定哪些因子是翻译所需的， HCV和登革病毒mRNA。这些研究将确定病毒mRNAs在多大程度上不同于 加帽/聚腺苷酸化的mRNA在其起始机制中的作用，并且可以鉴定治疗性的靶点。 这些病毒的感染。大量证据表明，eIF 3在细胞内起着重要的作用。 调节蛋白质合成和细胞增殖。与其他起始因子一样，eIF 3被磷酸化， 许多亚基蛋白。磷酸化位点将通过质谱法鉴定， 在翻译测定中测试磷酸化对功能的影响。变异体将是 构建并测试，其中磷酸化位点被丙氨酸取代以防止磷酸化，以及 用谷氨酸或天冬氨酸模拟磷酸化。这些实验旨在阐明 eIF 3的作用机制和磷酸化在启动过程中调节其活性的作用 加帽和IRES驱动的mRNA。这些结果可能解释了elF 3的功能障碍或失调如何可能 影响对细胞增殖的控制，导致细胞恶性化。