KINETIC DISSECTION OF EUKARYOTIC TRANSLATION INITIATION

描述(改编自申请人的摘要)：开始翻译 在真核生物中是一个极其复杂的过程，涉及at的作用。 至少26个非核糖体多肽，需要从 三磷酸腺苷和三磷酸鸟苷的水解酶。在启动过程结束时，一个 核糖体组装在mRNA的正确起始密码子上，具有 甲硫酰引发剂tRNA结合在其肽(P)位和第二位 结合在其受体(A)位的氨酰tRNA。在这个阶段，核糖体是 具有活性，并准备催化形成第一个多肽键。而当 启动过程中的许多步骤已经被描绘出来，并扮演了 在已建立的一些启动因素中，人们对 这一基本生物过程的分子力学： 平移机械各部件之间的相互作用和运动 催化和协调建立启蒙计划所需的每个步骤 复杂？ 这项提案概述了解剖 真核细胞体外翻译起始途径。这些方法将是 用于建立描述每个步骤的动力学和热力学框架 启动过程。这样的框架对于尝试 理解翻译启动的分子力学。这些技术 并将使用动力学和热力学框架来探索分子 信使核糖核酸的结构特征在基因表达中的作用 翻译的起始：5‘-7-甲基鸟苷帽结构， 3‘-聚(A)尾、5’-非翻译区的结构和序列 围绕着起始密码子。还描述了一些实验，以探索 启动潜在新步骤的结构和生化基础 过程，依赖GTP的80S核糖体复合体的激活。这些研究 这份提案中概述的内容将为多年的目标工作奠定基础 了解真核生物所需的每个事件的分子基础 翻译启蒙。

DESCRIPTION (adapted from applicant's abstract): The initiation of translation in eukaryotes is an extraordinarily complex process involving the action of at least 26 non-ribosomal polypeptides and requiring energy input from the hydrolysis of both ATP and GTP. At the end of the initiation process, a ribosome is assembled at the correct initiation codon of an mRNA with a methionyl initiator tRNA bound in its peptidyl (P) site and the second aminoacyl tRNA bound in its acceptor (A) site. At this stage the ribosome is active and is poised to catalyze the formation of the first peptide bond. While many of the steps in the initiation process have been delineated and the roles of some of the initiation factors established, little is known about the molecular mechanics of this fundamental biological process: how do the interactions among and motions of the components of the translational machinery catalyze and coordinate each of the steps required to build an initiation complex? This proposal outlines physical biochemical approaches for dissecting the pathway of eukaryotic translation initiation in vitro. These approaches will be used to establish a kinetic and thermodynamic framework describing each step in the initiation process. Such a framework will be crucial for attempts to understand the molecular mechanics of translation initiation. These techniques and the kinetic and thermodynamic framework will be used to probe the molecular roles of structural features of the mRNA that are known to be important for the initiation of translation: the 5'-7-methylguanosine cap structure, the 3'-poly(A) tail, structure in the 5'-untranslated region and the sequence surrounding the initiation codon. Experiments are also described to explore the structural and biochemical bases for a potential new step in the initiation process, a GTP-dependent activation of the 80S ribosomal complex. The studies outlined in this proposal will lay the foundation for many years of work aimed at understanding the molecular basis for each event required for eukaryotic translation initiation.