Kinetic Dissection of Eukaryotic Translation Initiation

描述 翻译起始是一个重要的生物学过程。它也是基因表达调控的关键点。真核翻译起始是抗肿瘤、抗病毒和抗真菌药物的潜在靶点。然而，由于缺乏关于翻译过程分子机制的信息，开发靶向翻译起始的药物的尝试将受到阻碍。 该提案描述了旨在理解真核蛋白质合成机制识别mRNA中翻译起始位点的分子机制的实验。这可以说是基因表达过程中最重要的遗传密码阅读，因为如果它出错，就会产生错误编码的蛋白质。在识别起始密码子后，信号被发送到中心G蛋白起始因子eIF 2以不可逆地水解其结合的GTP并将甲硫氨酰起始剂tRNA释放到小核糖体亚基的P位点。该事件是翻译启动的第一个承诺步骤;发生后，复合物必须在mRNA的该点继续启动，否则将中止该过程。因此，eIF 2对GTP的不可逆水解必须受到非常仔细的调节，这样它就不会发生在mRNA上的错误位置，而是在正确的位置非常迅速地发生。43 S-mRNA前起始复合物形成的分子机制、mRNA中起始密码子的识别以及eIF 2引发不可逆GTP水解将通过本提案中描述的实验来阐明。这项工作将采用重组酵母为基础的翻译起始系统。这些步骤所需的启动机制的每个关键组成部分的作用和机制将阐明通过热力学和动力学解剖的途径。这些研究还将使用许多突变形式的起始因子，这些起始因子在体内产生良好表征的表型，这些表型是由我们的合作者分离的。因此，这些研究将协同利用酵母遗传学和分子生物学的力量，在体外进行详细的生物物理和生物化学研究。

DESCRIPTION Translation initiation is a central biological process. It is also a key point in the regulation of gene expression. Eukaryotic translation initiation is a potential target of anticancer, antiviral and antifungal drugs. Attempts to develop drugs targeting translation initiation will be hampered, however, by the paucity of information about the molecular mechanics of this process. This proposal describes experiments aimed at understanding the molecular mechanisms underlying the recognition of the translation initiation site in an mRNA by the eukaryotic protein synthesis machinery. This is arguably the most important reading of the genetic code during gene expression because if it goes awry a miscoded protein will be produced. Upon recognition of the initiation codon, a signal is sent to the central G protein initiation factor eIF2 to irreversibly hydrolyze its bound GTP and release the methionyl initiator tRNA into the P site of the small ribosomal subunit. This event is the first committed step in translation initiation; after it happens the complex must proceed with initiation at that point on the mRNA or abort the process. Thus the irreversible hydrolysis of GTP by eIF2 must be regulated exquisitely carefully such that it does not happen at the wrong place on the mRNA but happens very rapidly at the right place. The molecular mechanics of the formation of the 43S-mRNA pre-initiation complex, its identification of the start codon in the mRNA, and the triggering of irreversible GTP hydrolysis by eIF2 will be elucidated by the experiments described in this proposal. The work will employ a reconstituted yeast-based translation initiation system. The roles and mechanisms of each of the key components of the initiation machinery required for these steps will be elucidated through a thermodynamic and kinetic dissection of the pathway. These studies will also use a number of mutant versions of initiation factors that produce well characterized phenotypes in vivo, which were isolated by our collaborators. These studies will thus synergistically harness the power of yeast genetics and molecular biology to the detailed biophysical and biochemical studies possible in vitro.