Imaging Protein Synthesis on the Ribosome using Single-Molecule FRET

描述（由申请人提供）：翻译是蛋白质合成的过程，通过普遍保守的机制发生，该机制对生命所有领域的基因表达至关重要。翻译在人类细胞中受到高度调节，翻译控制的丧失是癌细胞生长的关键决定因素。细菌中的蛋白质合成是用于对抗传染病的一系列临床重要抗生素的目标。然而，对这些化合物的耐药性越来越普遍。核糖体是细胞翻译器的主要组成部分，是调控的整合点。为了填补这一知识空白，核糖体功能和翻译保真度的分子机制将使用最先进的生物物理方法进行研究，包括单分子全内反射荧光和零模式波导成像技术。使用这些平台，第一个多维的，高空间和时间分辨率的距离测量不同的构象自由度将获得在元素和过程蛋白质合成反应。再加上在分子动力学模拟和冷冻电子显微镜领域的合作和互补努力，这些研究将揭示翻译机制中结构事件的顺序和时间，以及它们如何有助于驱动定向和高保真蛋白质合成。长期目标是建立一个定量框架，将核糖体中构象事件的微观速率常数与全球蛋白质合成联系起来。这将揭示新的光的速率决定的过程中的结构事件，以及翻译保真度的分子基础，并将提供重要的见解，了解核糖体调控的细胞机制和临床相关的小分子效应翻译的行动。所获得的结果的合成将提供关于精确聚焦的动态结构过程的新信息，该过程支撑翻译机制，并为探索在反应坐标期间发生的特定事件如何可能被靶向用于治疗目的提供平台。 公共卫生相关性：该研究的重点是将最先进的成像技术应用于基因表达的翻译控制研究。这种多步骤和高度调节的过程是生长、分化和肿瘤发生的核心，靶向翻译的化合物是用于治疗疾病的疗法库的核心组分。

DESCRIPTION (provided by applicant): Translation, the process of protein synthesis, occurs via a universally conserved mechanism that is central to gene expression in all domains of life. Translation is highly regulated in human cells and the loss of translation control is a key determinant of cancerous cell growth. Protein synthesis in bacteria is targeted by a broad array of clinically-important antibiotics that are used to combat infectious disease. However, resistance to these compounds is increasingly widespread. The ribosome is the principal component of the cellular translation apparatus and is the integration point for regulation. In order to fill this knowledge gap, the molecular mechanism of ribosome function and translational fidelity will be investigated using state-of-the-art biophysical methods, including single-molecule Total Internal Reflection Fluorescence and zero-mode waveguide imaging technologies. Using these platforms, the first multidimensional, high-spatial and -temporal resolution distance measurements of distinct conformational degrees of freedom will be obtained during both elemental and processive protein synthesis reactions. Together with collaborative and complementary efforts in the areas of molecular dynamics simulations and cryo-electron microscopy, these investigations will reveal the order and timing of structural events in translation machinery and how they contribute to driving directional and high-fidelity protein synthesis. The long-term goal is to establish a quantitative framework that relates the microscopic rate constants of conformational events in the ribosome to global protein synthesis. This will shed new light on the rate-determining structural events in the process as well as the molecular basis of translation fidelity, and will provide insights critical to understanding cellular mechanisms of ribosome regulation and the action of clinically-relevant small molecule effectors of translation. A synthesis of the results obtained will provide novel information about precisely focused, dynamic structural processes underpinning the translation mechanism and a platform for exploring how specific events that occur during the reaction coordinate may be targeted for therapeutic purpose. PUBLIC HEALTH RELEVANCE: The focus of the proposed research is to apply state-of-the-art imaging technologies to the study of translational control of gene expression. This multistep and highly regulated process is central to growth, differentiation and tumorigenesis and compounds targeting translation are central components of the arsenal of therapies for the treatment of disease.