KINETIC DISSECTION OF EUKARYOTIC TRANSLATION INITIATION

描述（改编自申请人摘要）：翻译的开始 是一个非常复杂的过程，涉及到 至少26个非核糖体多肽，并需要能量输入从 ATP和GTP的水解。在启动过程结束时， 核糖体在mRNA的正确起始密码子处组装， 甲硫氨酰引发剂tRNA结合在其肽基（P）位点， 氨酰tRNA结合在其受体（A）位点。在这个阶段，核糖体 具有活性并准备催化第一个肽键的形成。而 启动过程中的许多步骤已经被描述出来， 在已建立的一些起始因子中， 这一基本生物过程的分子力学： 平移机构各部件之间的相互作用和运动 促进和协调建立一个初始化所需的每个步骤 复杂吗 该提案概述了解剖的物理生物化学方法， 真核生物体外翻译起始途径。这些方法将 用于建立描述每个步骤的动力学和热力学框架， 启动过程。这样一个框架将是至关重要的尝试， 理解翻译起始的分子力学。这些技术 动力学和热力学框架将用于探测分子 mRNA的结构特征的作用，已知是重要的， 翻译的起始：5 '-7-甲基鸟苷帽结构， 3 '-poly（A）尾，5'-非翻译区的结构和序列 围绕着起始密码子。还描述了实验以探索 结构和生物化学基础的一个潜在的新的一步， 这是一个GTP依赖的80 S核糖体复合物激活过程。研究 该提案中概述的工作将为多年的工作奠定基础， 在理解真核生物所需的每个事件的分子基础， 翻译起始

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.