Imaging Protein Synthesis on the Ribosome using Single-Molecule FRET

 描述（由申请人提供）：细胞信使RNA翻译成不同的结构和功能蛋白质是生命所有领域基因表达的核心，是蛋白质组能力和多样性的关键通道。翻译的多步骤和高度调节的过程是由核糖体进行的，核糖体是一种两亚基的RNA-蛋白质组装体，由细菌中的70种不同的基因产物和人类中的80多种不同的基因产物组成。虽然翻译机制的结构和功能特征被认为在整个进化过程中是保守的，但靶向细菌翻译机制的小分子抗生素的治疗性给药仍然是对抗世界各地现有和新出现的传染病的关键保障。因此，人类的翻译机制和/或核糖体结构的关键方面必须与细菌不同。人类细胞中翻译控制的丧失与癌症生长有关，越来越多的知识表明癌细胞内的核糖体可能在物理和功能上不同。相应地，细菌和人类核糖体的共同和独特特征的知识提供了改善现有抗生素功效的潜力，开发了潜在的抗生素干预新方法，并有望为癌症治疗提供预防特异性策略。这些方面的进展需要定量描述细菌和人类翻译机制中的结构-功能关系以及翻译机制的动态方面，包括蛋白质合成过程中发生的核糖体组成和构象的时间依赖性变化。拟议的研究旨在阐明核糖体功能的分子基础，翻译保真度的起源以及使用一系列最先进的生物物理方法的抗生素作用的分子机制。这些方法包括单分子全内反射荧光成像和快速停止流动动力学测量，以及在分子动力学模拟和高分辨率结构测定方面的协作和互补努力。在这样做的过程中，我们的目标是描绘细菌和人类核糖体功能的定量动力学和结构模型，以定义关键的区别，这将为治疗感染性病原体和人类疾病的小分子干预提供新的机会。

 DESCRIPTION (provided by applicant): The translation of cellular messenger RNAs into distinct structural and functional proteins is central to gene expression in all domains of life an serves as a critical conduit for proteome capacity and diversity. The multistep and highly regulated process of translation is carried out by the ribosome, a two-subunit, RNA-protein assembly that is composed of 70 distinct gene products in bacteria and more than 80 distinct gene products in humans. Although the structural and functional features of the translation mechanism are thought to be conserved throughout evolution, the therapeutic administration of small-molecule antibiotics targeting the bacterial translation machinery continues to serve as a critical safeguard against existing and emerging infectious diseases around the world. Hence, key aspects of the translation mechanism and/or ribosome structure in humans must be distinct from bacteria. The loss of translation control in human cells is linked to cancerous growth and a growing body of knowledge suggests that ribosomes within cancer cells may be physically and functionally distinct. Correspondingly, knowledge of the common and distinct features of bacterial and human ribosomes offers the potential to enable improvements in the efficacies of existing antibiotics, the development of potentially novel means for antibiotic intervention and holds the promise of translation-specific strategies for cancer treatment. Progress on these fronts requires quantitative descriptions of structure-function relationships in the translation machinery of bacteria and humans as well as dynamic aspects of the translation mechanism, including time-dependent changes in ribosome composition and conformation that occur during processive protein synthesis reactions. The proposed research aims to elucidate the molecular basis of ribosome function, the origins of translational fidelity and the molecular mechanisms of antibiotic action using a battery of state-of-the-art biophysical approaches. The methods include single-molecule Total Internal Reflection Fluorescence imaging and rapid-stopped flow kinetics measurements, together with collaborative and complementary efforts in molecular dynamics simulation and high-resolution structure determination. In so doing, we aim to delineate quantitative kinetic and structural models of bacterial and human ribosome function to define key distinctions that will inform on new opportunities for small-molecule interventions for the treatment of infectious pathogens and human disease.