Ribosome Structure and Function

核糖体结构和功能

• 批准号: 9895828
• 负责人: HARRY F NOLLER
• 金额: $ 77.23万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA CRUZ
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9895828
• 关键词: Antibiotics; Biochemistry; Biophysics; Clinical; Coupled; Crystallization; Crystallography; Elongation Factor; Fluorescence Resonance Energy Transfer; Genes; Genetic; Genetic Diseases; Goals; Human; In Vitro; Knowledge; Link; Messenger RNA; Methods; Modeling; Molecular; Movement; Mutation; Peptide Elongation Factor G; Population; Positioning Attribute; Process; Property; Protein Biosynthesis; Proteins; Public Health; Research; Ribosomal Proteins; Ribosomal RNA; Ribosomes; Role; Structure; TRAP Complex; Testing; Time; Transfer RNA; Translating; Translations; Work; exhaustion; genetic information; mutant; public health relevance; reconstitution; single molecule

 DESCRIPTION (provided by applicant): We are entering a new and exciting era of ribosome research, in which the role of ribosome dynamics in protein synthesis is beginning to emerge. This proposal focuses on the role of the structural dynamics of the ribosome, and ribosomal RNA in particular, in the mechanism of translation. Our approach uses a combination of biochemistry, biophysics, genetics and crystallography. A major goal is to elucidate the mechanism of the coupled translocation of mRNA and tRNA. One strategy is to carry out an exhaustive screen for dominant-lethal mutations in elongation factor EF-G, which we predict will trap the ribosome in previously unobserved transient states of translocation. We then plan to crystallize these trapped complexes and solve their structures, to fill in gaps in our knowledge of the sub-steps of this fundamental process. To link these static, crystallographically-derived snapshots to ribosome dynamics, we will follow internal movements of the ribosome using FRET. We will take advantage of our ability to reconstitute active ribosomes containing fluorescent FRET pairs attached to specific positions, allowing us to follow ribosome dynamics in real time in both ensemble and single-molecule contexts. Mechanistic models derived from these studies can then be tested by creating pure populations of mutant ribosomes using methods that we have previously developed to target mutations to either rRNA or ribosomal proteins, and testing their functional properties in vitro.

 描述（由应用程序提供）：我们正在进入一个新的令人兴奋的核糖体研究时代，其中核糖体动力学在蛋白质合成中的作用开始出现。该建议的重点是核糖体的结构动力学，尤其是核糖体RNA的作用，在翻译机理中的作用。我们的方法结合了生物化学，生物物理学，遗传学和晶体学。一个主要目标是阐明mRNA和tRNA的耦合易位的机制。一种策略是对伸长因子EF-G中的主要致命突变进行详尽的筛选，我们预测，这将使核糖体陷入以前未观察到的易位瞬时状态。然后，我们计划结晶这些被困的复合物并解决它们的结构，以填补我们的知识中的空白 这个基本过程的子步骤。为了将这些静态的，晶体学衍生的快照与核糖体动力学联系起来，我们将使用FRET遵循核糖体的内部运动。我们将利用我们重建包含连接到特定位置的荧光帧对的活性核糖体的能力，从而使我们能够在集合和单分子上下文中实时遵循核糖体动力学。然后，可以通过使用我们以前开发的方法来靶向突变或核糖体蛋白并在体外测试其功能特性来测试从这些研究中得出的机械模型。