Kinetic Dissection of Eukaryotic Translation Initiation

翻译启动是一个重要的生物学过程。它也是基因调控中的一个关键点。 表情。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，开发针对翻译启动的药物的尝试将受到以下因素的阻碍 关于这一过程的分子力学的信息。 这项建议描述了旨在了解导致脑出血的分子机制的实验。 真核蛋白质合成机制对信使核糖核酸翻译起始点的识别。这是 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 就会产生编码错误的蛋白质。一旦识别了起始密码子，信号就被发送到中央G 蛋白起始因子elF2不可逆水解其结合的GTP并释放甲硫基启动子tRNA 插入到小核糖体亚基的P位。这一事件是翻译启动的第一步承诺； 在此之后，复合体必须在信使核糖核酸的那个点上继续启动，否则就中止这个过程。 因此，elF2对GTP的不可逆水解必须进行精细的调控，使其不会 在信使核糖核酸上的错误位置发生，但在正确的位置发生得非常快。分子 43S？m RNA预起始复合体的形成机制及其起始密码子的鉴定 以及elF2对GTP不可逆水解性的触发将通过实验来阐明。 本提案中所描述的。这项工作将采用一种重组的基于酵母的翻译启动系统。 这些所需的启动机制的每一个关键部件的作用和机制 这些步骤将通过对该途径的热力学和动力学剖析来阐明。这些研究将 还使用了许多启动因子的突变版本，这些突变版本在 是由我们的合作者分离出来的。因此，这些研究将协同利用 酵母菌遗传学和分子生物学能够在体外进行详细的生物物理和生化研究。

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 elF2 to irreversibly hydrolyze its bound GTP and release the methionyl initiator tRNA into the P site of the smallribosomal 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 elF2 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 elF2 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.