The role played by the RNAs and tRNAs in translation

将细胞中的遗传信息翻译成功能蛋白质是一种基本的、高度保守的 功能。核糖体是细菌中由三个大核糖核酸组成的两个亚单位的大分子复合体 (RRNAs)和50多种蛋白质，是这一复杂和协调过程的催化剂和框架 翻译的问题。理论上的考虑，生化，遗传和现在的结构证据表明 RRNAs在翻译过程中起着核心作用，包括氨酰tRNA负载、多肽键 MRNA的形成、转位：tRNA复合体和多肽的释放。我们的总体目标是定义 通过关注rRNA在其中每一个中所起的作用，介绍了翻译过程的分子细节 基本步骤。虽然结构信息为我们提供了可能发挥直接作用的候选区域 在催化和运动中，我们无法在缺乏生化的情况下定义它们的具体作用 解剖。此外，翻译周期的动态很难从静态视图中提取出来 这是我们从X射线结晶学和低温电子显微镜方法中获得的。该提案概述了一系列 实验重点是使用稳态前动力学分析来确定特定rRNA的贡献 翻译中不同步骤的元素。定点突变将针对关键区域和一个 活体标记系统将用于体外分离变异核糖体。停流 荧光将被用来监测tRNA结合、释放因子相互作用和易位，同时快速 将使用猝灭方法来研究GTP的水解率、多肽键的形成和多肽的形成 放手。还建议进行化学修饰实验和基因筛选，以帮助 确定rRNA中在翻译动态中起关键作用的其他区域。信息 从这类研究中获得的结果将有助于确定使其有效的当前抗生素的特征 并最终在设计新的治疗化合物类别中发挥作用。

Translation of the genetic information in the cell into functional proteins is an essential and highly conserved function. The ribosome, a two-subunit macromolecular complex, composed in bacteria of three large RNAs (rRNAs) and more than 50 proteins, is the catalyst and framework for the intricate and coordinated process of translation. Theoretical considerations, biochemical, genetic and now structural evidence indicate that the rRNAs play a central role in the processes of translation including aminoacyl tRNA loading, peptide bond formation, translocation of the mRNA:tRNA complex and peptide release. Our general goals are to define the molecular details of the translation process by focusing on the role played by the rRNAs in each of these basic steps. While structural information has provided us with candidates regions that may play direct roles in catalysis and movement, we are unable to define their specific role in the absence of biochemical dissection. Further, the dynamics of the translation cycle have been difficult to extract from the static views that we obtain from X-ray crystallography and cryoEM approaches. This proposal outlines a series of experiments focused on using pre-steady state kinetic analysis to define the contributions of specific rRNA elements to the different steps in translation. Site-directed mutagenesis will target critical regions and an in vivo tagging system will be used to allow for the isolation of variant ribosomes in vitro. Stopped-flow fluorescence will be used to monitor tRNA binding, release factor interactions and translocation while rapid quench approaches will be used to study the rates of GTP hydrolysis, peptide bond formation and peptide release. Chemical modification experiments and genetic screens are also proposed to assist in the identification of other regions of the rRNA that are critical in the dynamics of translation. Information obtained from such studies will help in defining the features of current antibiotics that make them effective and ultimately in the design of the new classes of therapeutic compounds.