The Role of Eukaryotic Initiation Factor (eIF) 4G in Initiation of Translation

helen MCB 9726958技术真核起始因子（eIF） 4G通过与核糖体募集mRNA的其他因子相互作用，协调蛋白质合成的限速步骤。eIF4G与帽结合蛋白eIF4E和atp酶/RNA解旋酶eIF4A结合形成eIF4F复合物。eIF4G也与eIF3结合，eIF3是43S核糖体起始前复合物的一个组成部分。本研究应用生化和分子遗传学方法来表征eIF4G'在翻译起始中的作用。这是通过在体外使用纯化组分（包括重组eIFs 4A、4B、4E和4G）对多种克隆mrna进行重组实验来实现的。为此目的，开发了灵敏的足印和足迹方法来绘制核糖体和mrna上的起始前复合物的位置。这项研究的意义在于它提高了我们在分子水平上对蛋白质合成的认识。2. 本研究旨在阐明蛋白质合成机制和调控的基本方面，特别是起始因子4G （eIF4G）在协调各种细胞成分的活动中所起的作用，这些细胞成分共同招募单个mrna以开始翻译。实验中，不同mrna上纯化的组分在翻译开始阶段在体外重组。各种蛋白质或mRNA成分被系统地替换为一系列变体，并在特定条件下（如生长停滞和有丝分裂）比较它们的效果。eIF4G中决定因素的重要性，使其能够非特异性地结合所有mrna，并与少数具有高特异性的mrna结合，被识别和表征。本研究选择了高亲和力结合eIF4G的病毒和细胞mrna，因为它们可能是eIF4E失活时翻译的模型。与eIF4G（另一种起始因子）相互作用的eIF4A的决定因素也在研究中。这种相互作用引导eIF4A解绕到mRNA上的特定位点，从而允许核糖体附着，核糖体是蛋白质合成的锚定机制。

Hellen MCB 9726958 1. Technical Eukaryotic initiation factor (eIF) 4G coordinates the rate-limiting step in protein synthesis by interacting with other factors in the recruitment of mRNA to ribosomes. eIF4G associates with the cap-binding protein eIF4E and the ATPase/RNA helicase eIF4A to form the eIF4F complex. eIF4G also binds to eIF3, a component of the 43S ribosomal preinitiation complex. This study applies biochemical and molecular genetic approaches to characterize the role of eIF4G' in initiation of translation. This is achieved through reconstitution experiments in vitro using purified components, which include recombinant eIFs 4A, 4B, 4E and 4G, on a variety of clone mRNAs. Sensitive toe-printing and footprinting methods to map the position of ribosomal and preinitiation complexes on mRNAs are developed for this purpose. The significance of this study lies in its advancing our knowledge of protein synthesis at the molecular level. 2. Non Technical This study is to elucidate fundamental aspects of the mechanism and regulation of protein synthesis, particularly the role of the initiation factor, 4G (eIF4G), in coordinating the activities of various cellular components that together recruit individual mRNAs for the onset of translation. Experimentally, purified components on different mRNAs are reconstituted in vitro at the beginning stage of translation. Various protein or mRNA components are systematically substituted with a series of variants, and their effects compared under specific conditions such as growth arrest and mitosis. The importance of determinants in eIF4G that enable it to bind to all mRNAs nonspecifically, and to a few with high specificity, are identified and characterized. Viral and cellular mRNAs that bind eIF4G with high affinity are chosen in this study, since they may be models for translation when eIF4E is inactive. Also being studied are determinants of eIF4A's which interact with eIF4G, another initiation factor. This interaction directs the unwinding of eIF4A to a specific site on the mRNA to permit the attachment of ribosomes, the anchorage machinery for protein synthesis.