Kinetic Dissection of Eukaryotic Translation Initiation

描述（由申请人提供）：翻译起始的控制在从发育到学习和记忆到细胞死亡的广泛的正常生物过程中是重要的。翻译起始的不正确执行和控制涉及多种人类疾病的病因学，包括病毒感染、神经变性和癌症。为了理解翻译起始是如何被控制的，以及这种控制是如何被打破的，我们必须首先理解正常过程的基本分子机制。本申请中提出的实验将加深我们对真核生物翻译起始的分子基础的理解。这项工作将雇用一个完全重组的S。酿酒酵母翻译起始系统，并将涉及与美国国立卫生研究院/NICHD的Alan Hinnebusch实验室的密切合作。这些研究将解决我们对真核生物翻译起始的理解中的两个主要空白。第一个目标集中于mRNA中的起始密码子被核糖体前起始复合物识别的机制。起始密码子的识别是遗传密码最重要的解读之一，因为它为信息的解码设定了框架。不正确的起始位点选择导致产生错误编码的、潜在有毒的蛋白质。拟议的研究将阐明前起始复合物中的信息传递途径，从起始密码子和起始tRNA反密码子之间的三个碱基对的初始形成到下游不可逆事件，这些事件使复合物在信息上的选定点完成起始。我们的研究将集中在启动子tRNA本身和启动因子eIF 5在这一过程中发挥的作用。大量的数据表明，这两个组成部分是我们理解起始密码子识别的分子基础中缺失的环节。第二个主要目的是剖析mRNA募集到前起始复合物的分子机制，这是蛋白质合成中的关键事件，也是调控的关键靶点。在这个资助周期中，mRNA募集的研究将集中在大的异源多聚体因子eIF 3和ssRNA结合蛋白eIF 4 B的作用机制上。初步工作表明，这两个因素在这一过程中发挥着核心作用，但目前对它们如何发挥作用知之甚少。

DESCRIPTION (provided by applicant): Control of translation initiation is important in a wide range of normal biological processes, from development to learning and memory to cell death. Improper execution and control of translation initiation are involved in the etiology of a variety of human diseases, including viral infection, neurodegeneration and cancer. In order to understand how translation initiation is controlled, and how this control can break down, we must first understand the underlying molecular mechanics of the normal process. The experiments proposed in this application will deepen our understanding of the molecular basis of translation initiation in eukaryotes. The work will employ a fully reconstituted S. cerevisiae translation initiation system and will involve close collaboration with the laboratory of Alan Hinnebusch at the NIH/NICHD. The studies proposed will address two major gaps in our understanding of eukaryotic translation initiation. The first aim focuses on the mechanism through which the start codon in an mRNA is recognized by the ribosomal pre-initiation complex. Recognition of the start codon is one of the most important readings of the genetic code because it sets the frame for decoding of the message. Improper start site selection results in production of a miscoded, potentially toxic, protein. The proposed studies will elucidate the pathways of information transfer within the pre-initiation complex, from initial formation of three base pairs between the start codon and initiator tRNA anticodon to the downstream irreversible events that commit the complex to finishing initiation at the selected point on the message. Our studies will focus on the roles that the initiator tRNA itself and the initiation factor eIF5 play in this process. A significant amount of data indicates that these two components are missing links in our understanding of the molecular basis of start codon recognition. The second major aim is to dissect the molecular mechanics of mRNA recruitment to the pre-initiation complex, which is a critical event in protein synthesis and a key target of regulation. In this grant cycle, studies of mRNA recruitment will focus on the mechanisms of action of the large, heteromultimeric factor eIF3 and the ssRNA binding protein eIF4B. Preliminary work indicates central roles for both factors in this process, yet currently little is known about how they function.