Ribosome Structure and Function

核糖体结构和功能

• 批准号: 10597704
• 负责人: HARRY F NOLLER
• 金额: $ 76.6万
• 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 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10597704
• 关键词: Address; Antibiotics; Antitubercular Agents; Antitubercular Antibiotics; Biochemistry; Clinical; Collaborations; Complex; Computing Methodologies; Coronavirus; Coupled; Cryoelectron Microscopy; Development; Elongation Factor; Fluorescence Resonance Energy Transfer; Genetic; HIV; Head; Human; Label; Laboratories; Measurement; Messenger RNA; Methods; Molecular; Molecular Conformation; Movement; Mutation; Pathogenicity; Peptide Elongation Factor G; Protein Biosynthesis; Proteins; Public Health; RNA Viruses; Reading Frames; Research; Ribosomal RNA; Ribosomes; Rotation; Site; Structure; Transfer RNA; Translating; Viomycin; Work; exhaust; genetic information; helicase; insight; laser tweezer; model design; novel; novel strategies; optic tweezer; preservation; single molecule; single-molecule FRET; structural biology

Project Summary This project focuses on understanding the molecular mechanisms underlying the coupled translocation of mRNA and tRNAs during protein synthesis. It includes the important related problems of how the translational reading frame is preserved (or shifted) and how the ribosomal helicase unwinds structured mRNAs. Our laboratory uniquely uses a combination of biochemistry, structural biology, genetics, FRET and computational methods to address these challenging problems. We are also extending our approaches to include single-molecule optical tweezer methods, in collaboration with the Bustamante laboratory (UC Berkeley) and single- molecule FRET, in collaboration with the Ermolenko laboratory (Univ. of Rochester), as well as cryo-electron microscopy, in collaboration with the Chiu laboratory (Stanford/SLAC). In previous studies, we have determined the structures of trapped translocation intermediates, which have provided unexpected insights into how the movements of mRNA and tRNA through the ribosome are coupled to large- and small-scale conformational changes in the structure of the ribosome itself. We then created FRET pairs that allowed us to correlate intersubunit rotation, movement of the L1 stalk and rotation of the 30S subunit head domain with movements of mRNA and tRNA. We plan to extend this search to discover new intermediate states. Having exhausted previous strategies for trapping translocation intermediates, we will use a new approach which exploits a set of dominant-lethal mutations in all five structural domains of elongation factor EF-G that we expect will block translocation at different steps. Development of a novel fluorescent labeling approach that will allow site-specific labeling of FRET pairs directly to ribosomal RNA will overcome technical barriers to single-molecule studies of ribosome dynamics, including studies using simultaneous measurement of molecular forces and FRET changes in the ribosome, in collaboration with the Bustamante group. Finally, we have designed model structured mRNAs that will provide the basis for studying the mechanism of the mRNA helicase and for determination of the structures of translocation complexes stalled in the act of encountering and unwinding an mRNA helix.

项目摘要 该项目的重点是理解该项目的分子机制 蛋白质合成过程中mRNA和TRNA的耦合易位。它包括重要的 有关如何保留（或移动）的转化阅读框架的相关问题以及如何 核糖体解旋酶松开结构的mRNA。我们的实验室独特地结合了 生物化学，结构生物学，遗传学，FRET和计算方法来解决这些问题 具有挑战性的问题。我们还在扩展我们的方法以包括单分子光学 镊子方法与Bustamante实验室（UC Berkeley）和单一合作 分子方格，与Ermolenko实验室（罗切斯特大学）合作， 冷冻电子显微镜与CHIU实验室（Stanford/SLAC）合作。 在先前的研究中，我们确定了被困易位的结构 中间体为mRNA和mRNA的运动提供了意外的见解 通过核糖体的tRNA耦合到大型和小规模的构象变化 核糖体本身的结构。然后，我们创建了Fret对，使我们能够关联 subunit旋转，L1茎的运动以及30S亚基头域的旋转 mRNA和tRNA的运动。我们计划扩展此搜索以发现新的中间体 国家。耗尽了以前的策略来捕获易位中间体，我们将 使用一种在所有五个结构中利用一组主导致命突变的新方法 我们期望的伸长因子EF-G的域将在不同步骤中阻止易位。 开发一种新型的荧光标签方法，该方法将允许特定地点标记 直接与核糖体RNA成对的粮食将克服单分子的技术障碍 核糖体动力学的研究，包括同时测量分子的研究 与Bustamante Group合作，核糖体的部队和FRET变化。最后， 我们设计了模型结构的mRNA，这将为研究 mRNA解旋酶的机理和确定易位结构 在遇到和放松mRNA螺旋的行为中，建筑群停滞了。