Imaging protein synthesis on the ribosome using single-molecule FRET

使用单分子 FRET 对核糖体上的蛋白质合成进行成像

• 批准号: 7496916
• 负责人: Scott C Blanchard
• 金额: $ 30.99万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-29 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7496916
• 关键词: Address; Affect; Amino Acid Sequence; Antibiotics; Arts; Binding; Biochemical; Biochemical Genetics; Biological Process; Cancerous; Cell physiology; Cells; Codon Nucleotides; Coupling; Cryoelectron Microscopy; Crystallography; Data; Docking; EEF1A1 gene; Elements; Elongation Factor; Engineering; Enzymes; Event; Fluorescence; Foundations; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Hydrolysis; Image; Investigation; Joints; Kinetics; Measurement; Measures; Mediating; Messenger RNA; Methodology; Methods; Microscopic; Microscopy; Modeling; Monitor; Motion; Movement; Mutation; Nature; Normal Cell; Peptide Elongation Factor G; Peptide Elongation Factor Tu; Peptide Sequence Determination; Peptidyltransferase; Play; Process; Protein Biosynthesis; Proteins; RNA; Rate; Regulation; Research; Research Personnel; Resolution; Ribosomes; Ricin; Role; Screening procedure; Site; Techniques; Testing; Therapeutic; Time; Transfer RNA; Translational Regulation; Translations; cell growth; fluorophore; human EEF1A1 protein; infectious disease treatment; insight; instrument; molecular assembly/self assembly; molecular dynamics; novel; particle; programs; reconstitution; research study; single molecule; single-molecule FRET; stem; structural biology; tool; translation factor; Address; Affect; Amino Acid Sequence; Antibiotics; Arts; Binding; Biochemical; Biochemical Genetics; Biological Process; Cancerous; Cell physiology; Cells; Codon Nucleotides; Coupling; Cryoelectron Microscopy; Crystallography; Data; Docking; EEF1A1 gene; Elements; Elongation Factor; Engineering; Enzymes; Event; Fluorescence; Foundations; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Hydrolysis; Image; Investigation; Joints; Kinetics; Measurement; Measures; Mediating; Messenger RNA; Methodology; Methods; Microscopic; Microscopy; Modeling; Monitor; Motion; Movement; Mutation; Nature; Normal Cell; Peptide Elongation Factor G; Peptide Elongation Factor Tu; Peptide Sequence Determination; Peptidyltransferase; Play; Process; Protein Biosynthesis; Proteins; RNA; Rate; Regulation; Research; Research Personnel; Resolution; Ribosomes; Ricin; Role; Screening procedure; Site; Techniques; Testing; Therapeutic; Time; Transfer RNA; Translational Regulation; Translations; cell growth; fluorophore; human EEF1A1 protein; infectious disease treatment; insight; instrument; molecular assembly/self assembly; molecular dynamics; novel; particle; programs; reconstitution; research study; single molecule; single-molecule FRET; stem; structural biology; tool; translation factor

DESCRIPTION (provided by applicant): The principal component of the translation machinery, and the integration point for regulation of protein synthesis is the ribosome, a two-subunit, RNA-protein enzyme. In concert with RNA and protein translation factors, the ribosome converts messenger RNA (mRNA) into a specific protein sequence. Translation is highly regulated in normal cell physiology; loss of translation control is a key determinant of cell growth in the cancerous state. Translation is also targeted by a broad array of clinically useful therapeutic compounds important for the treatment of infectious diseases. Recent breakthroughs in structural biology have yielded spectacular snapshot images of the ribosome and its components. These data provide the foundation for a much needed physical framework upon which genetic, biochemical and biophysical investigations of ribosome function must be reconciled. To this end, we propose to study the mechanism of tRNA selection and translocation processes on the single-molecule scale. The hypothesis central to the proposed research is that translation fidelity stems from conformational changes within the ribosome that are triggered by its interaction with tRNA and translation factors. Using state-of-the-art Total Internal Reflection Fluorescence techniques we will extract the global and microscopic rate constants of the dynamic structural changes of the translation machinery from high-spatial and -time resolution distance measurements. These data, unobtainable in ensemble studies, will aid in the elucidation of the basic translation mechanism. They will serve as a platform for the investigation of translational regulation mechanisms within the cell, and for screening novel compounds which affect specific aspects of ribosome function. To test our hypothesis, we will probe the tRNA selection and translocation mechanisms using single-molecule FRET (smFRET) as a tool to make direct measurements of the dynamic, structural processes within the functioning ribosome in a reconstituted translation extract. Specifically, we propose: AIM 1] Measure the order and timing of tRNA motions on the ribosome during Elongation Factor-Tu (EF-Tu)-mediated tRNA selection and Elongation Factor-G (EF-G)-dependent translocation. AIM 2] Determine how movements and conformational changes of EF- Tu, and EF-G relate to the mechanisms of aa-tRNA selection and translocation, respectively. AIM 3] Delineate the influence of conformational changes of the ribosome during protein synthesis on the motions of tRNA during tRNA selection and translocation. The instruments and methodologies developed through this research may also be applicable to investigations of other molecular assemblies where compositional and conformational processes play a role in biological function.

描述（由申请人提供）：翻译机械的主要成分，蛋白质合成调节的集成点是核糖体，一种两亚基的RNA蛋白质酶。与RNA和蛋白质翻译因子一致，核糖体将信使RNA（mRNA）转化为特定的蛋白质序列。在正常细胞生理学中，翻译受到高度调节。翻译控制的丧失是癌状态细胞生长的关键决定因素。翻译也是针对多种临床上有用的治疗化合物，对治疗传染病很重要。结构生物学的最新突破产生了核糖体及其成分的壮观快照图像。这些数据为急需的物理框架提供了基础，必须对核糖体功能进行遗传，生化和生物物理研究。为此，我们建议在单分子量表上研究tRNA选择和易位过程的机制。拟议研究的核心假设是翻译保真度源于核糖体内的构象变化，这些变化是由于其与tRNA和翻译因子的相互作用而触发的。使用最先进的内部反射荧光技术，我们将从高空间和 - 时间分辨率距离测量值中提取转换机械动态结构变化的全局和显微镜速率常数。这些数据在整体研究中无法获得，将有助于阐明基本的翻译机制。它们将作为研究细胞内翻译调节机制的平台，并筛选影响核糖体功能特定方面的新型化合物。为了检验我们的假设，我们将使用单分子FRET（SMFRET）作为一种工具来探测tRNA选择和易位机制，以在重构翻译提取物中直接测量功能性核糖体中的动态结构过程。具体而言，我们提出：目标1]测量在伸长因子-TU（EF-TU）介导的tRNA选择和伸长延伸因子G（EF-G）依赖性转运期间，核糖体对核糖体上tRNA运动的顺序和时间。目标2]确定EFTU的运动和构象变化以及EF-G分别与AA-TRNA选择和易位的机制有关。 AIM 3]描述蛋白质合成过程中核糖体构象变化的影响对tRNA选择和易位过程中tRNA运动的影响。通过这项研究开发的工具和方法也可能适用于对组成和构象过程在生物学功能中起作用的其他分子组件的研究。