REGULATORS OF TRANSLATION ELONGATION FACTOR 1 ALPHA

翻译延伸因子1α(EF-1α)是一种核心蛋白 参与向核糖体运送氨基酰tRNAs并确保 在蛋白质合成过程中加入适当的氨基酸。 虽然这种蛋白质极其丰富，但细胞需要调节 细胞正常生长所需的EF-1α活性或水平 EF-α表达不当导致细胞转化 并出现在许多癌症中。此外，活动中的变化 EF-1α的表达发生在衰老和延长寿命的过程中。这样做的目的是 这个项目的目的是了解监管高效和 准确的翻译。有许多不同的机制来确保 准确和高效地翻译一个mRNA，这是显而易见的 机制集中在EF-1α的作用上。的核心作用 EF-1α的伸长及其与蛋白质和RNA的相互作用 在这个过程中，调节其活性是一个非常重要的因素。我们的 以前的研究支持这样一种假设，即翻译中的变化 携带EF-1α突变的细胞的保真度是许多因素的结果 对体内基因表达的不同影响。当前型号的 延伸率预测这些包括由以下因素引起的间接影响 活动EF-1αx GTP池的变化，从而占用 核糖体的A位。然而，EF-1α也直接影响 这一过程通过AA-tRNA递送到核糖体， 同源密码子-反密码子相互作用的测定，以及 动力学校对所需的GTP水解酶。的用法 酿酒酵母允许对翻译进行分析 体内和体外的伸长周期。本提案描述了一种 一套预测或设计用来分析突变的生化研究 EF-1α对精确基因的直接和间接作用 表情。野生型和突变型EF-1α的纯化与分析 蛋白质在翻译延伸过程中的部分反应会 确定体内翻译和生长表型的原因 在这些突变中。此外，这些信息被用来应用 识别和表征调节因素的遗传学方法 EF-1α活性；鸟嘌呤核苷酸交换因子(EF-1β) 和GTP酶激活因子(核糖体)。这一分析将 还要确定影响或调节尖端伸长的新因素。 这种协调一致的方法是扩大我们理解的独特方法 在许多依赖EF-1α的步骤中，准确和 高效的基因表达。

Translation Elongation Factor 1alpha (EF-1alpha) is a central protein involved in delivering aminoacyl-tRNAs to the ribosome and assuring incorporation of the appropriate amino acid during protein synthesis. While this protein is extremely abundant, the cell needs to modulate the activity or levels of EF-1alpha for normal cellular growth since inappropriate expression of EF-alpha results in transformation of cell lines and occurs in many carcinomas. Further, changes in the activity of EF-1alpha occur during aging and alter longevity. The goal of this project is to understand the mechanisms that regulate efficient and accurate translation. There are many different mechanisms to assure the accurate and efficient translation of an mRNA, and it is clear these mechanisms converge on the action of EF-1alpha. The central role of EF-1alpha in elongation and its interactions with proteins and RNAs that modulate its activity are a very important factor in this process. Our previous studies support the hypothesis that changes in translational fidelity in cells harboring mutations in EF-1alpha are a result of many different effects on gene expression in vivo. Current models of elongation predict these include indirect effects that result from changes in the pool of active EF-1alpha x GTP and thus the occupancy of the A-site of the ribosome. However, EF-1alpha also directly affects this process through the presentation of aa-tRNA to the ribosome, the determination of cognate codon-anticodon interactions, and the hydrolysis of GTP required for kinetic proofreading. The use of the yeast Saccharomyces cerevisiae allows analysis of the translation elongation cycle both in vivo and in vitro. This proposal describes a set of biochemical studies of mutations predicted or designed to analyze both direct and indirect effects of EF-1alpha on accurate gene expression. Purification and analysis of wild-type and mutant EF-1alpha proteins in the partial reactions of translation elongation will determine the causes of the in vivo translation and growth phenotypes of these mutations. Furthermore, this information is utilized to apply genetic approaches to identify and characterize factors that modulate EF-1alpha activity; the guanine nucleotide exchange factor (EF-1Beta) and the GTPase activating factor (the ribosome). This analysis will also identify novel factors that affect or regulate acurate elongation. This coordinated approach is a unique method to extend our understanding of the many EF-1alpha dependent steps required for accurate and efficient gene expression.