Kinetic Dissection of Eukaryotic Translation Initiation

描述（由申请人提供）：翻译起始的控制在广泛的正常生物过程中是重要的，从发育到学习和记忆再到细胞死亡。翻译起始的不当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破坏的，我们必须首先了解正常过程的潜在分子力学。本应用程序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。这项工作将采用一个完全重组的cerevisiae翻译起始系统，并将与美国国立卫生研究院/美国国立卫生研究院的Alan Hinnebusch实验室密切合作。提出的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差距。第一个目的是研究核糖体起始前复合物识别mRNA起始密码子的机制。起始密码子的识别是对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。不当的起始位点选择导致产生错误编码的、潜在有毒的蛋白质。所提出的研究将阐明预起始复合物内的信息传递途径，从起始密码子和启动tRNA反密码子之间的三个碱基对的初始形成到下游的不可逆事件，这些不可逆事件使复合物在信息上的选定点完成起始。我们的研究将重点关注启动物tRNA本身和启动因子eIF5在这一过程中所起的作用。大量的数据表明，这两个组成部分是我们理解启动密码子识别的分子基础的缺失环节。第二个主要目的是剖析mRNA招募到起始前复合体的分子机制，起始前复合体是蛋白质合成的关键事件，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聚因子eIF3和ssRNA结合蛋白eIF4B的作用机制上。初步研究表明，这两个因素在这一过程中起着核心作用，但目前对它们的作用知之甚少。

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. PUBLIC HEALTH RELEVANCE: The final stage of the expression of genes is the production of the proteins that each gene encodes. When this process - called "protein synthesis" or "translation" - is performed incorrectly a variety of diseases can result, including neurodegeneration, anemia and cancer. Our studies will increase our understanding of how translation normally works, which in turn will increase our understanding of how it can break down and result in diseases.