Experimentally Guided Ribosomal RNA Modeling

实验引导的核糖体 RNA 建模

• 批准号: 8136343
• 负责人: JOACHIM FRANK
• 金额: $ 27.56万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8136343
• 关键词: Address; Amino Acyl Transfer RNA; Antibiotics; Anticodon; Area; Biochemical; Biochemistry; Cells; Chemistry; Classification; Codon Nucleotides; Collaborations; Complex; Cryoelectron Microscopy; Czech Republic; DNA Sequence Rearrangement; Data; Data Set; Devices; Discrimination; Drug resistance; EEF1A1 gene; Equilibrium; Freezing; Grant; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Heterogeneity; Hybrids; Hydrolysis; Image; Imagery; In Vitro; Kinetics; Life; Maps; Messenger RNA; Methods; Microfluidics; Modeling; Molecular; Molecular Conformation; Motion; Movement; Mutation; National Center for Research Resources; Peptidyltransferase; Phase; Population; Positioning Attribute; Process; Protein Biosynthesis; Publishing; Research; Resolution; Resources; Ribosomal RNA; Ribosomes; Roentgen Rays; Ruthenium Ben; Sampling; Seminal; Site; Sorting - Cell Movement; Specimen; Structure; Sweden; Techniques; Technology; Testing; Time; Time Study; Transfer RNA; United States National Institutes of Health; Universities; Work; base; combat; density; design; flexibility; interest; millisecond; molecular dynamics; nanofabrication; novel; particle; public health relevance; reconstruction; research study; simulation; single-molecule FRET; time use

DESCRIPTION (provided by applicant): This proposal seeks continued support for exploration of structure and function of the ribosome actively engaged in protein synthesis, by cryo-electron microscopy and single-particle reconstruction. Several seminal discoveries regarding the dynamics of this process have been made in pursuit of this approach; among these the ratchet motion during mRNA-tRNA translocation and the spring-like deformation of the aminoacyl-tRNA as it enters the ribosome. The emphasis of the proposed studies is twofold: to characterize suitably stabilized transition states during both decoding and translocation at high resolution, and to study the time course of both processes using time-resolved cryo-electron microscopy (ms range) of pre-equilibrium samples. A novel monolithic microfluidic mixing and spraying device developed at the NCRR/NIH RVBC Resource at the Wadsworth Center in Albany will be tested and used in collaboration with the Resource, and the technology will be later transferred to the Columbia lab. Samples will be obtained from collaborators Drs. Rachel Green, Johns Hopkins University and Mans Ehrenberg, Uppsala University. Additional collaborations are in the areas of classification of heterogeneous samples and Molecular Dynamics simulations. Density maps when obtained at sufficient resolution will be analyzed by flexible fitting and interpreted in the rich context of structural, kinetics, single-molecule FRET, and biochemical data. PUBLIC HEALTH RELEVANCE: The ribosome performs protein synthesis in all cells. The research proposed will further the understanding of its functional dynamics, and thus contribute to a fundamental understanding of all life processes. Specifically, understanding the function of bacterial ribosomes is furthering our ability to combat drug resistance.

描述（由申请人提供）：该提案寻求继续支持通过冷冻电子显微镜和单粒子重建来探索积极参与蛋白质合成的核糖体的结构和功能。在追求这种方法的过程中，已经取得了一些关于这一过程动力学的开创性发现；其中包括 mRNA-tRNA 易位过程中的棘轮运动以及氨酰基-tRNA 进入核糖体时的弹簧状变形。拟议研究的重点是双重的：以高分辨率表征解码和易位过程中适当稳定的过渡态，并使用预平衡样品的时间分辨冷冻电子显微镜（毫秒范围）研究这两个过程的时间过程。位于奥尔巴尼沃兹沃斯中心的 NCRR/NIH RVBC 资源中心开发的新型整体式微流体混合和喷涂装置将与该资源中心合作进行测试和使用，该技术随后将转移到哥伦比亚实验室。样品将从合作者 Drs 处获得。雷切尔·格林（Rachel Green），约翰·霍普金斯大学和曼斯·埃伦伯格（Mans Ehrenberg），乌普萨拉大学。其他合作涉及异质样品分类和分子动力学模拟领域。以足够的分辨率获得的密度图将通过灵活的拟合进行分析，并在结构、动力学、单分子 FRET 和生化数据的丰富背景下进行解释。 公共健康相关性：核糖体在所有细胞中进行蛋白质合成。所提出的研究将进一步了解其功能动力学，从而有助于对所有生命过程的基本理解。具体来说，了解细菌核糖体的功能正在进一步增强我们对抗耐药性的能力。