Kinetic Dissection of Eukaryotic Translation Initiation

描述 翻译启动是一个重要的生物学过程。它也是基因表达调控中的一个关键点。真核翻译起始是抗癌、抗病毒和抗真菌药物的潜在靶点。然而，由于缺乏关于这一过程的分子机制的信息，开发针对翻译起始的药物的尝试将受到阻碍。 这项建议描述了旨在了解真核蛋白质合成机制识别mRNA中翻译起始点的分子机制的实验。这可以说是基因表达过程中对遗传密码最重要的读取，因为如果它出错，就会产生错误编码的蛋白质。一旦识别了起始密码子，信号就被发送到中心G蛋白起始因子eIF2，使其结合的GTP不可逆地水解，并将甲硫基启动子tRNA释放到小核糖体亚基的P位。这个事件是翻译启动的第一个承诺步骤；在它发生后，复合体必须在mRNA上的那个点继续启动，否则就放弃这个过程。因此，eIF2对GTP的不可逆水解性必须受到严格的调控，这样才不会发生在mRNA的错误位置，而是迅速地发生在正确的位置。43S-mRNA预起始复合体的形成、其在mRNA中起始密码子的鉴定以及eIF2触发不可逆GTP水解的分子机制将通过本建议中描述的实验来阐明。这项工作将采用一种重组的基于酵母的翻译启动系统。这些步骤所需的启动机制的每个关键组件的作用和机制将通过对该途径的热力学和动力学剖析来阐明。这些研究还将使用一些突变版本的启动因子，这些突变版本在体内产生特征良好的表型，这些表型是我们的合作者分离出来的。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化研究。

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.