The role played by the RNAs and tRNAs in translation

RNA 和 tRNA 在翻译中发挥的作用

• 批准号: 7529208
• 负责人: RACHEL GREEN
• 金额: $ 25.48万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7529208
• 关键词: Active Sites; Amino Acyl Transfer RNA; Antibiotics; Bacteria; Binding; Biochemical; Biochemical Genetics; Biogenesis; Catalysis; Cells; Chemicals; Complex; Cryoelectron Microscopy; Dissection; Elements; Genetic; Genetic Code; Genetic Screening; Genetic Translation; Goals; Guanosine Triphosphate; Hydrolysis; In Vitro; Indium; Kinetics; Macromolecular Complexes; Messenger RNA; Modification; Molecular; Monitor; Movement; Mutate; Mutation; Nucleotides; Peptides; Play; Process; Proteins; RNA; Research; Research Personnel; Resolution; Ribosomal RNA; Ribosomes; Role; Scheme; Series; Signal Transduction Pathway; Site-Directed Mutagenesis; Specific qualifier value; Structure; System; Therapeutic; Transfer RNA; Translation Process; Translations; Variant; Work; X-Ray Crystallography; catalyst; defined contribution; design; in vivo; interest; molecular mechanics; novel; polypeptide; programs; release factor; research study; stopped-flow fluorescence; tool

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.

将细胞中的遗传信息翻译成功能蛋白质是一个重要的和高度保守的过程。 功能核糖体是细菌中由三种大分子RNA组成的双亚基大分子复合体 核糖体RNA（rRNA）和50多种蛋白质，是复杂和协调过程的催化剂和框架 的翻译。理论上的考虑，生物化学，遗传学和现在的结构证据表明， rRNA在翻译过程中起着重要的作用，包括氨酰tRNA的装载、肽键的形成、 mRNA：tRNA复合物的形成、易位和肽释放。我们的总体目标是定义 翻译过程的分子细节，通过关注rRNA在每个翻译过程中所起的作用， 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，我们无法在缺乏生物化学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 我们从X射线晶体学和冷冻电镜方法中获得的。该提案概述了一系列 实验集中于使用前稳态动力学分析来确定特定rRNA的贡献， 翻译中的不同步骤。定点突变将靶向关键区域， 体内标记系统将用于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， 淬灭方法将用于研究GTP水解，肽键形成和肽 release.化学修饰实验和遗传筛选也被提出来协助 鉴定在翻译动力学中至关重要的rRNA的其他区域。信息 从这些研究中获得的信息将有助于确定当前抗生素的有效特征 并最终用于新型治疗化合物的设计。