EIF3 STRUCTURE /FUNCTION IN INITIATION AT IRESS

该项目的主要目标是了解人类翻译启动因子elF3如何对 对病毒mRNAs，尤其是丙型肝炎病毒mRNAs的启动机制和调控 通过核糖体结合到内部进入位点(IRES)来启动翻译。ElF3的结构，一个因素 由12个不同的亚基组成，将通过分离在 杆状病毒感染的昆虫细胞。将定义elF3及其亚复合体与丙型肝炎病毒IRES的结合 从数量上讲。依赖于纯化的elF3的高活性部分分级哺乳动物细胞裂解物系统 将用(5-珠蛋白mRNA)构建。这个系统将被用来定义elF3及其亚复合体的作用 在丙型肝炎病毒信使核糖核酸的翻译中。还将构建体外翻译系统， 利用所有纯化的启动因子，以定义翻译所需的因子 丙型肝炎病毒和登革病毒mRNAs。这些研究将确定病毒mRNAs与 帽/多腺化的mRNAs在其启动机制中的作用，并可能识别治疗靶点 这些病毒感染的干预。大量证据表明elF3起着重要的作用 调节蛋白质合成和细胞增殖。像其他启动因子一样，elF3被磷酸化。 大量的亚基蛋白质。磷酸化的位置将通过质谱学鉴定，而 磷酸化对功能的影响将在翻译试验中进行测试。突变形式将是 构建和测试，用丙氨酸取代磷酸化位点以防止磷酸化，以及 用谷氨酸或天冬氨酸模拟磷酸化。这些实验旨在阐明 ElF3的作用机制及磷酸化在调控其活性中的作用 封顶和IRES驱动的mRNAs。结果可能解释elF3的功能失调或失调可能 影响细胞增殖的控制，导致细胞恶性转化。

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.