Single Molecule Dynamics of mRNA Translation

mRNA 翻译的单分子动力学

• 批准号: 7526959
• 负责人: BARRY S. COOPERMAN
• 金额: $ 29.86万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7526959
• 关键词: Address; Affect; Amino Acyl Transfer RNA; Benchmarking; Binding; Cell physiology; Cells; Chloramphenicol O-Acetyltransferase; Code; Codon Nucleotides; Collection; Computer software; Coupling; Dihydrofolate Reductase; EEF1A1 gene; Elements; Escherichia coli; Evaluation; Fluorescence; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Genetic Translation; Goals; Height; Indium; Kinetics; Label; Length; Location; Messenger RNA; Methods; Microscope; Modeling; Molecular Profiling; Monitor; Mutation; Peptide Elongation Factor Tu; Peptides; Photobleaching; Play; Procedures; Process; Protein Biosynthesis; Proteins; Public Health; Quantum Dots; Rate; Reagent; Recovery; Regulation; Relative (related person); Research; Ribosomes; Role; Silent Mutation; Site; Speed; Structure; Suggestion; System; Time; Transfer RNA; Translating; Translations; Work; design; helicase; human EEF1A1 protein; image processing; improved; insight; instrumentation; interest; mutant; protein folding; single molecule; single-molecule FRET; stem

DESCRIPTION (provided by applicant): Our goal is exploit the power of single molecule observation to elucidate the mechanism by which specific sequences within mRNA modulate the rate of translation by E. coli ribosomes, through use of an approach coupling Total Internal Reflection Fluorescence Microscopy (TIRFM) with Fluorescence Resonance Energy Transfer (FRET). In our approach, fluorescent groups are introduced in the ribosome that, by FRET interaction with fluorescently-labeled tRNAs, allow initial aminoacyl-tRNA binding to the ribosomal A-site, tRNA translocation to the P-site, and release of discharged tRNA from the E-site to be monitored on single ribosomes in real time. This approach will provide a detailed, continuous kinetic profile of mRNA translation during continuous elongation, providing unique insights into the regulation of translation rates in protein synthesis that are important for cell function. We will determine kinetic profiles for expression of i) short model mRNAs containing known pausing elements regulating translation; ii) complete mRNAs coding for full proteins; and iii) designed mutations of such mRNAs that permit rigorous evaluation of the effects of pausing elements, singly or in groups, in the context of full protein synthesis. Such determinations will allow new understanding of the roles such pauses play in biologically important processes and providing suggestions for optimizing cell-free protein synthesis systems. Our specific aims are to: 1. Determine translation profiles for model mRNAs. We will determine translation kinetic profiles for model mRNAs incorporating known pausing elements (rare codons, downstream mRNA 2o structure, upstream nascent peptides) either one at a time or in tandem. The information obtained will quantify effects of such elements on translation and elucidate the mechanism of the intrinsic ribosomal helicase. 2. Determine translation rates for full-length mRNAs. We will determine translation kinetic profiles for full length mRNAs and specifically designed mutants of such mRNAs in order to determine how surrounding context influences the effect of a given pausing element or group of pausing elements on translation rate, beginning with the mRNAs coding for E. coli dihydrofolate reductase (DHFR) and chloramphenicol acetyltransferase (CATIII). 3. Optimize the reagents employed in the TIRFM-FRET approach. The principal improvements over currently available reagents will be directed toward i. reducing background from fluorescently-labeled tRNA by derivatizing EF-Tu with a fluorescence quencher; ii. synthesizing a larger variety of fluorescent tRNAs; and iii. labeling ribosomes with quantum dots for increased stability toward photobleaching. 4. Optimize the apparatus and methods needed for the TIRFM-FRET approach. Several improvements to the apparatus, software and procedures will be accomplished to enable collection of long kinetic sequences from the onset of elongation, with high fidelity and minimum perturbation by photobleaching. The image processing software used to quantify single molecule FRET pairs and their efficiencies will be improved, optimized and statistically validated. PUBLIC HEALTH RELEVANCE: Three major consequences will flow from our work. First, we will be able to examine the effects of known translational pausing elements in the context of complete protein chain expression, and to determine if new elements and synergistic effects can be identified. Second, we will be able to systematically explore the role translational pausing plays in the integration of protein synthesis and cellular function, focusing on such issues as the tradeoff between speed and accuracy in translation at functionally crucial residues, and the possible coupling of translational pausing and co-translational protein folding. Third, we will be able to provide important information with respect to optimizing cell-free protein translation systems.

描述（由申请人提供）：我们的目标是利用单分子观察的力量，通过使用全反射荧光显微镜（TIRFM）和荧光共振能量转移（FRET）相结合的方法，阐明mRNA中特定序列调节大肠杆菌核糖体翻译速率的机制。在我们的方法中，在核糖体中引入荧光基团，通过FRET与荧光标记的tRNA相互作用，允许初始氨基酰基tRNA与核糖体a位点结合，tRNA易位到p位点，以及从e位点释放的tRNA在单个核糖体上实时监测。这种方法将提供mRNA在持续延伸过程中翻译的详细、连续的动力学概况，为蛋白质合成中翻译率的调节提供独特的见解，这对细胞功能很重要。我们将确定i)含有调节翻译的已知暂停元件的短模型mrna的表达动力学谱；ii)编码完整蛋白的完整mrna；iii)设计这些mrna的突变，以便在全蛋白质合成的背景下，对单个或成组暂停元件的作用进行严格评估。这样的决定将允许新的理解这种暂停在生物学重要过程中发挥的作用，并为优化无细胞蛋白质合成系统提供建议。我们的具体目标是：1。确定模型mrna的翻译谱。我们将确定包含已知暂停元件（稀有密码子，下游mRNA 20结构，上游新生肽）的模型mRNA的翻译动力学特征，无论是一次还是串联。所获得的信息将量化这些元素对翻译的影响，并阐明内在核糖体解旋酶的机制。2. 确定全长mrna的翻译速率。我们将从编码大肠杆菌二氢叶酸还原酶（DHFR）和氯霉素乙酰转移酶（CATIII）的mrna开始，确定全长mrna和专门设计的mrna突变体的翻译动力学特征，以确定周围环境如何影响给定暂停元件或暂停元件组对翻译率的影响。3. 优化TIRFM-FRET方法中使用的试剂。目前可用试剂的主要改进将是：1 .通过荧光猝灭剂衍生化EF-Tu来减少荧光标记tRNA的背景；2。合成更多种类的荧光trna；ⅲ。用量子点标记核糖体增加光漂白稳定性。4. 优化TIRFM-FRET方法所需的设备和方法。将完成对仪器、软件和程序的若干改进，以实现从伸长开始的长动力学序列的收集，具有高保真度和最小的光漂白扰动。用于量化单分子FRET对及其效率的图像处理软件将得到改进、优化和统计验证。公共卫生相关性：我们的工作将产生三个主要后果。首先，我们将能够在完整蛋白链表达的背景下检查已知的翻译暂停元件的作用，并确定是否可以识别新的元件和协同效应。其次，我们将能够系统地探索翻译停顿在蛋白质合成和细胞功能整合中的作用，重点关注功能关键残基翻译速度和准确性之间的权衡，以及翻译停顿和共翻译蛋白质折叠的可能耦合等问题。第三，我们将能够提供关于优化无细胞蛋白翻译系统的重要信息。