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和蛋白质翻译因子一起将信使RNA（mRNA）转化为特定的蛋白质序列。翻译在正常细胞生理学中受到高度调节;翻译控制的丧失是癌状态下细胞生长的关键决定因素。翻译也被广泛的临床上有用的治疗性化合物靶向，这些化合物对于治疗感染性疾病很重要。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壮观快照图像。这些数据提供了一个非常需要的物理框架的基础上，核糖体功能的遗传，生物化学和生物物理研究必须协调。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的构象变化，这些变化是由核糖体与tRNA和翻译因子的相互作用引发的。使用国家的最先进的全内反射荧光技术，我们将提取全球和微观速率常数的动态结构变化的翻译机器从高空间和时间分辨率的距离测量。这些数据，无法获得的合奏研究，将有助于阐明的基本翻译机制。它们将作为研究细胞内翻译调节机制以及筛选影响核糖体功能特定方面的新型化合物的平台。为了验证我们的假设，我们将使用单分子FRET（smFRET）作为直接测量重构翻译提取物中功能核糖体内的动态结构过程的工具来探测tRNA选择和易位机制。具体而言，我们建议：[目的1]测定在延伸因子Tu（EF-Tu）介导的tRNA选择和延伸因子G（EF-G）依赖的转运过程中tRNA在核糖体上运动的顺序和时间。[目的2]研究EF- Tu和EF-G的运动和构象变化与aa-tRNA选择和转位机制的关系。[目的3]阐明蛋白质合成过程中核糖体构象变化对tRNA选择和转运过程中运动的影响。通过这项研究开发的仪器和方法也可能适用于其他分子组装的组成和构象过程中发挥作用的生物功能的调查。

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.