Single Molecule Dynamics of mRNA Translation

描述(申请人提供)：我们的目标是利用单分子观察的力量，通过使用全内反射荧光显微镜(TIRFM)和荧光共振能量转移(FRET)相结合的方法，阐明mRNA中的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tRNAs相互作用，允许初始氨酰tRNA结合到核糖体A位，tRNA易位到P位，以及从E位释放的tRNA在单个核糖体上实时监测。该方法将提供连续伸长过程中mRNA翻译的详细、连续的动力学分布，为蛋白质合成中对细胞功能至关重要的翻译速率的调节提供独特的见解。我们将确定i)包含调节翻译的已知暂停元件的短模型mRNAs；ii)编码完整蛋白质的完整mRNAs；以及iii)此类mRNAs的设计突变，允许在完整蛋白质合成的背景下严格评估暂停元件的影响。这样的测定将使人们对这种停顿在生物重要过程中所起的作用有新的理解，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的翻译配置文件。我们将确定包含已知暂停元件(稀有密码子、下游mRNA2O结构、上游新生多肽)的模型mRNAs的翻译动力学图谱，要么一次一个，要么串联。所获得的信息将量化这些元件对翻译的影响，并阐明内在核糖体螺旋酶的机制。2.确定全长mRNAs的翻译速率。我们将确定全长mRNAs和这些mRNAs的特殊设计的突变体的翻译动力学图谱，以便确定周围环境如何影响给定的暂停元件或一组暂停元件对翻译速率的影响，首先是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mRNAs。3.优化TIRFM-FRET方法中使用的试剂。对现有试剂的主要改进将针对：1.通过用荧光猝灭剂衍生EF-Tu来降低荧光标记的tRNA的背景；合成更多种类的荧光tRNA；以及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。将完成对设备、软件和程序的几项改进，以便能够以高保真度和通过光漂白最小的扰动收集从伸长开始的长动力学序列。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。与公共卫生相关：我们的工作将产生三个主要后果。首先，我们将能够在完整的蛋白质链表达的背景下检查已知的翻译暂停元件的影响，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暂停在蛋白质合成和细胞功能整合中所起的作用，重点放在功能关键残基翻译的速度和准确性之间的权衡，以及翻译暂停和共翻译蛋白质折叠可能的耦合等问题。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。

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.