Imaging protein synthesis on the ribosome using single-molecule FRET

描述(申请人提供)：翻译机器的主要组成部分，调节蛋白质合成的整合点是核糖体，一种双亚基的RNA-蛋白质酶。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。翻译在正常的细胞生理学中受到高度调控；翻译控制的丧失是细胞在癌症状态下生长的关键决定因素。翻译也是一系列临床上有用的治疗化合物的目标，这些化合物对治疗传染病很重要。最近结构生物学的突破产生了核糖体及其组成部分的壮观快照图像。这些数据为迫切需要的物理框架提供了基础，必须在该框架上协调对核糖体功能的遗传、生化和生物物理研究。为此，我们建议在单分子水平上研究tRNA选择和转位过程的机制。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。使用最先进的全内反射荧光技术，我们将从高空间和时间分辨率的距离测量中提取平移机械动态结构变化的全局和微观速率常数。这些在系综研究中无法获得的数据，将有助于阐明基本的翻译机制。它们将作为一个平台，研究细胞内的翻译调控机制，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)作为工具来探索tRNA选择和易位机制，以直接测量重组翻译提取物中功能核糖体内的动态、结构过程。具体地说，我们提出：目的1]在延伸因子-Tu(EF-Tu)介导的tRNA选择和延伸因子-G(EF-G)依赖的易位过程中，测量tRNA在核糖体上运动的顺序和时间。目的]确定EF-Tu和EF-G的运动和构象变化分别与AA-tRNA选择和易位机制有关。[目的]阐明蛋白质合成过程中核糖体构象变化对tRNA选择和转位过程中tRNA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组成和构象过程在生物功能中发挥作用的其他分子组装体的研究。

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.