The role played by the RNAs and tRNAs in translation

描述（由申请人提供）：将细胞中的遗传信息翻译成功能性蛋白质是一种必需且高度保守的功能。核糖体是一种由两个亚基组成的大分子复合物，在细菌中由三种大RNA（rRNA）和50多种蛋白质组成，是复杂和协调的翻译过程的催化剂和框架。理论上的考虑，生物化学，遗传学和现在的结构证据表明，rRNA在翻译过程中起着核心作用，包括氨酰tRNA加载，肽键形成，mRNA：tRNA复合物的易位和肽释放。我们的总体目标是通过关注rRNA在每个基本步骤中所起的作用来定义翻译过程的分子细节。虽然结构信息为我们提供了可能在催化和运动中发挥直接作用的候选区域，但在没有生化解剖的情况下，我们无法确定它们的具体作用。此外，翻译周期的动态已经很难从我们从X射线晶体学和cryoEM方法获得的静态视图中提取。该提案概述了一系列实验，重点是使用前稳态动力学分析来定义特定rRNA元件对翻译中不同步骤的贡献。定点诱变将靶向关键区域，并且将使用体内标记系统以允许在体外分离变体核糖体。停流荧光将用于监测tRNA结合、释放因子相互作用和易位，而快速淬灭方法将用于研究GTP水解、肽键形成和肽释放的速率。化学修饰实验和遗传筛选也被提出来帮助识别在翻译动力学中至关重要的rRNA的其他区域。从这些研究中获得的信息将有助于确定当前抗生素的特征，使其有效，并最终设计新的治疗化合物。

DESCRIPTION (provided by applicant): 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.