Single Molecule Dynamics of mRNA Translation

mRNA 翻译的单分子动力学

• 批准号: 8127776
• 负责人: BARRY S. COOPERMAN
• 金额: $ 30.03万
• 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 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8127776
• 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; Health; Height; Indium; Kinetics; Label; Length; Location; Messenger RNA; Methods; Microscope; Modeling; Molecular Profiling; Monitor; Mutation; Peptides; Photobleaching; Play; Procedures; Process; Protein Biosynthesis; Proteins; Quantum Dots; Reagent; Recovery; Regulation; Relative (related person); Research; Ribosomes; Role; Silent Mutation; Site; Speed; Structure; Suggestion; System; Time; Transfer RNA; Translating; Translations; Work; design; helicase; 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来阐明mRNA中的特定序列通过使用方法偶联的总内反射荧光显微镜（TIRFM）与荧光谐振能量传递（Fret）通过方法耦合总内反射荧光显微镜（TIRFM）来调节大肠杆菌核糖体的翻译速率。在我们的方法中，在核糖体中引入荧光基团，通过与荧光标记的TRNA相互作用，允许初始的氨基酰基-TRNA与核糖体A位点结合，将TRNA转移到PITE，并释放从E-Site中释放出从E-Site中释放出e-LANNA，实际上是在单个核心体上监测的E-Site。这种方法将在连续伸长过程中提供mRNA翻译的详细，连续的动力学曲线，从而为蛋白质合成中翻译速率的调节提供独特的见解，这对细胞功能很重要。我们将确定表达的动力学曲线i）简短模型mRNA，其中包含已知的暂停元素调节翻译的元素； ii）完整编码全蛋白的mRNA； iii）设计了这种mRNA的突变，这些突变允许在完整的蛋白质合成的背景下单独或组中对暂停元件的影响进行严格评估。这种确定将使对这种暂停在生物学上重要的过程中所起的作用有了新的了解，并提供了优化无细胞蛋白质合成系统的建议。我们的具体目的是：1。确定模型mRNA的翻译曲线。我们将一次或一次串联确定模型mRNA的翻译动力学轮廓。获得的信息将量化此类元素对翻译的影响，并阐明固有核糖体解旋酶的机理。 2。确定全长mRNA的翻译速率。我们将确定全长mRNA和专门设计的此类mRNA突变体的翻译动力学曲线，以确定周围环境如何影响给定的暂停元素或暂停元素对翻译速率的效果，从编码大肠杆菌二氢叶酸盐还原酶（DHFR）的mRNA元素开始，并构成了氯甲基苯甲酸酯乙酰基乙二醇（Catiyltransyltransii）。 3。优化TIRFM-FRET方法中使用的试剂。对当前可用试剂的主要改进将针对i。通过用荧光淬火器衍生EF-TU从荧光标记的tRNA中降低背景； ii。合成较大的荧光TRNA；和iii。用量子点标记核糖体，以增加对光漂白的稳定性。 4.优化TIRFM-FRET方法所需的设备和方法。将对设备，软件和程序进行一些改进，从而从伸长率发作中收集长动动力学序列，并通过光漂白，并具有高忠诚度和最小的扰动。用于量化单分子FRET对及其效率的图像处理软件将得到提高，优化和统计验证。公共卫生相关性：我们的工作将带来三个重大后果。首先，我们将能够在完整的蛋白质链表达中检查已知的翻译暂停元件的影响，并确定是否可以鉴定出新的元素和协同作用。其次，我们将能够系统地探索蛋白质合成和细胞功能的整合中的转化暂停作用，重点介绍诸如功能上关键的残基的速度和准确性之间的折衷，以及翻译暂停和共同传播蛋白质折叠的可能耦合。第三，我们将能够提供有关优化无细胞蛋白质翻译系统的重要信息。