Imaging protein synthesis on the ribosome using single-molecule FRET

描述（由申请人提供）：翻译机制的主要组成部分和调节蛋白质合成的整合点是核糖体，一种双亚基rna -蛋白酶。核糖体与RNA和蛋白质翻译因子协同作用，将信使RNA （mRNA）转化为特定的蛋白质序列。翻译在正常细胞生理中受到高度调控；在癌变状态下，翻译控制的丧失是细胞生长的关键决定因素。翻译也是针对广泛的临床有用的治疗化合物，对治疗传染病很重要。结构生物学的最新突破已经产生了核糖体及其组成部分的惊人快照图像。这些数据为核糖体功能的遗传、生化和生物物理研究提供了一个急需的物理框架基础。为此，我们建议在单分子尺度上研究tRNA选择和易位过程的机制。这项研究的核心假设是，翻译保真度源于核糖体内部的构象变化，这种变化是由核糖体与tRNA和翻译因子的相互作用引发的。利用最先进的全反射荧光技术，我们将从高空间和时间分辨率的距离测量中提取翻译机器动态结构变化的全局和微观速率常数。这些在集合研究中无法获得的数据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化合物。为了验证我们的假设，我们将使用单分子FRET （smFRET）作为工具来探测tRNA选择和易位机制，以直接测量重建翻译提取物中功能核糖体内的动态结构过程。在伸长因子- tu （EF-Tu）介导的tRNA选择和伸长因子- g （EF-G）依赖的易位过程中，测量核糖体上tRNA运动的顺序和时间。AIM 2]确定EF- Tu和EF- g的运动和构象变化分别与aa-tRNA选择和易位机制的关系。[3]描述蛋白质合成过程中核糖体的构象变化对tRNA选择和易位过程中tRNA运动的影响。通过本研究开发的仪器和方法也可以应用于其他分子组合的研究，其中组成和构象过程在生物功能中起作用。

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.