Kinetic Dissection of Eukaryotic Translation Initiation

描述(由申请人提供)：翻译启动的控制在从发育到学习和记忆再到细胞死亡的广泛的正常生物过程中是重要的。翻译启动的不当执行和控制与多种人类疾病的病因学有关，包括病毒感染、神经退行性变和癌症。为了了解翻译启动是如何控制的，以及这种控制如何被打破，我们必须首先了解正常过程的潜在分子力学。本申请中提出的实验将加深我们对真核生物翻译启动的分子基础的理解。这项工作将使用一个完全重组的酿酒酵母翻译启动系统，并将涉及与美国国立卫生研究院/NICHD的艾伦·辛内布施的实验室密切合作。所提出的研究将解决我们对真核翻译启动的理解中的两个主要差距。第一个目的集中在核糖体前起始复合体识别mRNA中的起始密码子的机制上。对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。不正确的起始位置选择会导致产生一种编码错误的、潜在有毒的蛋白质。拟议的研究将阐明预引发复合体内的信息传递途径，从起始密码子和启动子tRNA反密码子之间最初形成三个碱基对到下游的不可逆事件，这些不可逆事件使复合体在信息的选定点处完成启动。我们的研究将集中在启动子tRNA本身和启动因子eIF5在这一过程中所起的作用。大量数据表明，在我们对起始密码子识别的分子基础的理解中，这两个组成部分缺少联系。第二个主要目的是剖析mRNA募集到前起始复合体的分子机制，这是蛋白质合成中的一个关键事件，也是调节的关键目标。在这个赠款周期中，对信使核糖核酸募集的研究将集中在大的异源多聚体因子eIF3和单链RNA结合蛋白eIF4B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了核心作用，但目前对它们如何发挥作用知之甚少。 与公共卫生相关：基因表达的最后阶段是每个基因编码的蛋白质的产生。当这一过程--称为“蛋白质合成”或“翻译”--执行不当时，可能会导致多种疾病，包括神经变性、贫血和癌症。我们的研究将增加我们对翻译正常工作方式的理解，这反过来又将增加我们对翻译如何破坏并导致疾病的理解。

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.