EIF3 STRUCTURE /FUNCTION IN INITIATION AT IRESS

IRESS 启动时的 EIF3 结构/功能

• 批准号: 7883199
• 负责人: JOHN W HERSHEY
• 金额: $ 15.37万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7883199
• 关键词: Affect; Alanine; Aspartate; Baculoviruses; Binding; Binding Sites; Cell Line; Cell Proliferation; Cells; Complex; Dengue; Dengue Virus; Eukaryotic Initiation Factor-3; Flavivirus; Genetic Translation; Globin; Glutamates; Goals; Hepatitis C virus; Human; In Vitro; Insecta; Internal Ribosome Entry Site; Malignant Neoplasms; Mammalian Cell; Mass Spectrum Analysis; Messenger RNA; Methods; Modification; Peptide Initiation Factors; Peptide Mapping; Phosphorylation; Phosphorylation Site; Play; Poly A; Protein Biosynthesis; Protein Subunits; Regulation; Research; Ribosomes; Role; Site; Structure; System; Testing; Therapeutic Intervention; Translating; Translations; Viral; Virus; Virus Diseases; design; in vitro testing; in vivo; mutant; prevent; programs; research study; translation assay

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ELF3如何贡献 启动机制和病毒mRNA的调节，尤其是丙型肝炎病毒（HCV）mRNA 这会通过核糖体结合到内部入口位点（IRES）来启动翻译。 ELF3的结构，一个因素 包括12个非相同亚基，通过隔离在 杆状病毒感染的昆虫细胞。将定义ELF3及其子复合物与HCV IRES的结合 定量。一个高度活跃的部分分离的哺乳动物细胞裂解物系统，取决于纯化的ELF3 将使用（5-珠蛋白mRNA。该系统用于定义ELF3及其子复合物的作用 在HCV mRNA的翻译中。还将构建一个体外翻译系统 利用所有纯化的启动因子，以定义翻译需要哪些因素 HCV和登革热病毒mRNA。这些研究将确定病毒mRNA与 在其启动机制中限制/聚腺苷酸化的mRNA，并可以识别治疗靶标 这些病毒对感染的干预。大量证据表明ELF3起重要作用 在调节蛋白质合成和细胞增殖方面。与其他起始因子一样，Elf3在 许多亚基蛋白。磷酸化的位点将通过质谱法确定，并且 磷酸化对功能的影响将在翻译测定中测试。突变形式将是 构造和测试，用磷酸化位点用丙氨酸取代以防止磷酸化，并且 与谷氨酸或天冬氨酸一起模拟磷酸化。实验旨在阐明 ELF3的作用机理以及磷酸化在调节其活性开始期间的作用 封顶和IRES驱动的mRNA。结果可能会解释ELF3的故障或不调如何 影响细胞增殖的控制，导致凯尔特恶性肿瘤。