Analysis of accurate ribosomal translocation

准确核糖体易位分析

• 批准号: 10389406
• 负责人: Kurt L Fredrick
• 金额: $ 7.23万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10389406
• 关键词: 3-Dimensional; Accounting; Address; Amino Acids; Amino Acyl Transfer RNA; Antibiotics; Bacteria; Biogenesis; Biological Assay; Cells; Chemicals; Complex; Cryoelectron Microscopy; Cytoplasm; Defect; Development; Elements; Enzymes; Escherichia coli; Eukaryotic Cell; Fluorescence Resonance Energy Transfer; Genetic Transcription; Goals; Growth; Guanosine Triphosphate Phosphohydrolases; Hereditary Disease; In Vitro; Individual; Inherited; Investigation; Ionic Strengths; Kinetics; Lead; Link; Malignant Neoplasms; Maps; Masks; Mass Spectrum Analysis; Measures; Medical; Messenger RNA; Modern Medicine; Modification; Molecular; Molecular Chaperones; Monitor; Multi-Drug Resistance; Nucleic Acids; Organism; Pathway interactions; Peptide Elongation Factor G; Physiology; Play; Prevention; Process; Proteins; Pseudouridine; Quality Control; RNA; RNA-Binding Proteins; Reporting; Ribosomal RNA; Ribosomes; Role; Sedimentation process; Seminal; Shapes; Shunt Device; Structure; Supplementation; System; Temperature; Testing; Translating; Translation Initiation; Translations; Uridine; Work; Yeasts; antimicrobial drug; base; cell assembly; cell growth; cell type; cold temperature; combat; emerging antibiotic resistance; experimental study; helicase; human disease; insight; kethoxal; novel; paralogous gene; particle; pathogen; premature; prevent; rRNA Precursor; reaction rate; reconstitution; ribosome profiling; self assembly

PROJECT SUMMARY (UNCHANGED) In all organisms, proteins are synthesized by ribosomes, large two-subunit enzymes that use aminoacyl-tRNA substrates to translate messenger RNA. Each ribosome is composed of several large RNA molecules (rRNAs) and more than 50 distinct proteins (r proteins), with rRNA accounting for around two-thirds of the overall mass. Seminal studies by the Nomura and Nierhaus groups showed that each ribosomal subunit can be reconstituted in vitro from its purified components, and hence the ribosome is fundamentally capable of self-assembly. However, such self-assembly is slow, inefficient, and requires non-physiological conditions. In the cell, numerous auxiliary proteins (termed assembly factors or AFs) facilitate the assembly process. Presumably, these AFs prevent and/or resolve low-energy intermediates (e.g., rRNA folding traps). But how these AFs act remains largely unknown, even in the simplest bacterial system. The long-term goal of the proposed work is to understand how rapid ribosome assembly is achieved in the cell. Aim 1. The GTPase BipA is a paralog of EF-G that plays some unclear role in 50S biogenesis. Cells lacking BipA grow poorly at low temperature and accumulate particles that represent a novel pre-50S (~40S) intermediate. The proposed work will characterize the structure of this intermediate, using chemical probing and cryo-electron microscopy approaches. The findings may define an intrinsic rRNA folding issue in 50S assembly and reveal the normal role of BipA. Aim 2. A growing body of evidence suggests that late-state 30S assembly occurs in the context of the 70S ribosome. The proposed work will investigate whether 30S biogenesis in bacteria includes a “test drive,” a translation-like cycle of quality control, as has been reported in eukaryotic cells. The findings may reveal an unappreciated functional link between ribosome assembly and translation initiation in bacteria. Aim 3. There currently exists no convenient assay to directly monitor 30S assembly in vitro. A FRET-based assay will be developed and used to investigate the roles of AFs, precursor rRNA elements, initiation components, and concurrent transcription on the rate of 30S assembly. Cellular components and/or parameters critical for rapid 30S biogenesis may be revealed and their mechanisms elucidated. Ribosomes are a main target of antibiotics, and defects in ribosome biogenesis cause many inherited human diseases (termed ribosomopathies). Insight gained by this project may ultimately lead to the development of novel antimicrobial drugs and/or treatments for one or more hereditary diseases.

项目摘要（不变） 在所有生物体中，蛋白质都是由核糖体合成的，使用氨基酰基-TRNA的大型两亚基酶 底物转换Messenger RNA。每个核糖体由几个大RNA分子（RRNA）组成 以及50多种不同的蛋白质（R蛋白），rRNA占整体质量的三分之二。 Nomura和Nierhaus组的开创性研究表明，每个核糖体亚基都可以重组 从其纯化的成分体外，因此核糖体从根本上具有自组装。 但是，这种自组装缓慢，效率低下，需要非生理条件。在细胞中， 许多辅助蛋白（称为装配因子或AFS）有助于组装过程。想必， 这些AFS预防和/或解决低能中间体（例如，rRNA折叠陷阱）。但是这些AFS如何行动 即使在最简单的细菌系统中，也基本上未知。拟议工作的长期目标是 了解细胞中核糖体组装的快速。 AIM1。GTPaseBIPA是EF-G的旁系同源物，在50S生物发生中起着不清楚的作用。缺乏细胞 BIPA在低温和代表新型前50年代（〜40s）的丙烯酸颗粒下生长较差 中间的。拟议的工作将使用化学探测来表征该中间体的结构 和冷冻电子显微镜方法。这些发现可能定义了50年代的内在rRNA折叠问题 组装并揭示BIPA的正常作用。 目标2。越来越多的证据表明，30年代后期的组装发生在70年代的背景下 核糖体。拟议的工作将调查细菌中30年代的生物发生是否包括“测试驱动”，A 正如真核细胞中报道的，质量控制的翻译样周期。调查结果可能会揭示 核糖体组装与细菌翻译起始之间的未批准的功能联系。 AIM 3。目前没有方便的测定方法可以直接在体外直接监视30S组件。基于货物的 测定将开发并用于研究AFS，前体rRNA元素的作用，启动 组件和30S组件速率的并发转录。细胞组件和/或 可以揭示对快速30S生物发生至关重要的参数，并阐明其机制。 核糖体是抗生素的主要靶标，核糖体生物发生缺陷导致许多遗传人类 疾病（称为核糖体病）。该项目获得的洞察力最终可能导致 一种或多种神经疾病的新型抗菌药物和/或治疗。