Imaging Protein Synthesis on the Ribosome using Single-Molecule FRET

项目摘要： 蛋白质合成及其在细胞中的调节机制决定了细胞的多样性和能力 蛋白质组。这种调控的中心整合点是核糖体：一个由两个亚单位组成的百万单位 RNA-蛋白质组装。突显了翻译和核糖体对调控的精致敏感性， 已知的大多数抗生素不是失调就是阻断核糖体功能。相应地，描述了 分子细节中的蛋白质合成机制有可能对基因表达的范式产生影响 关于控制和如何应对新出现的和具有抗药性的病原体的全球健康威胁。因为失去了 翻译控制是癌症的一个标志，对蛋白质合成机制的深入了解也 为目前缺乏的人类疾病治疗提供有针对性的治疗战略。 对支配翻译机制的结构-功能关系的研究主要是 使用传统的集合方法在细菌中进行。这类研究表明， 翻译是由信使核糖核酸(信使核糖核酸)合成蛋白质，称为延伸，是翻译最多的时间 密集且通常以毒品为目标。他们还发现，核糖体的伸长是瞬间发生的 通过一系列有序的事件与特定的细胞组件相互作用，其中每个事件的解码 MRNA密码子伴随着核糖体内的大规模构象变化和相互作用的因素， 以及核糖体及其mRNA和转移RNA(TRNA)底物之间的相互作用。需要大量的 同质化的材料阻碍了对人类翻译机制的类似研究。因此， 单细胞生物与哺乳动物之间翻译机制的保守性和差异性 决定抗生素特异性的分子基础在很大程度上仍然模糊不清。在这里，我们寻求 描述细菌和人类蛋白质合成的共同和不同特征--及其翻译 健康和癌症人体细胞的作用机制：1)提高现有抗生素的效力；2] 制定新的抗生素干预策略；以及3]探索不受控制的靶向治疗的可能性 增殖细胞生长和转移性扩散。我们将通过建立数量、结构和动力来实现这一点 使用集成电池的细菌和人类伸长的基本步骤的框架 生物物理方法，包括单分子荧光成像和最先进的低温电子 显微镜。我们的合作研究将描绘出构象事件的顺序和时间 细菌和人类伸长循环中的支撑保真度以及结构和机械差异 这决定了针对这些过程的临床相关抗生素的有效性。这些洞察力将会使 阐明翻译控制是如何实现的，揭示原子分辨率描述药物对细菌和 人类核糖体，并为新干预措施提供机会，以提高其疗效和效力 感染病原体和人类疾病的临床治疗。

PROJECT ABSTRACT: The mechanism of protein synthesis and its regulation in the cell determines the diversity and capacity of the proteome. The central integration point for this regulatory control is the ribosome: a two-subunit, megadalton RNA-protein assembly. Highlighting the exquisite sensitivity of translation and the ribosome to regulation, the majority of known antibiotics either dysregulate or block ribosome function. Correspondingly, delineation of the protein synthesis mechanism in molecular detail has the potential to inform on paradigms of gene expression control and on how to combat the global health threat of emerging and drug resistant pathogens. As the loss of translation control is a hallmark of cancer, a deeper understanding of the protein synthesis mechanism also holds the promise of targeted therapeutic strategies for human disease treatments that are currently lacking. Investigations into structure-function relationships governing the translation mechanism have been principally conducted in bacteria using traditional ensemble methods. Such studies have revealed that the phase of translation in which protein is synthesized from messenger RNA (mRNA), termed elongation, is the most time intensive and commonly drug-targeted. They have also discerned that elongation entails the ribosome transiently interacting with specific cellular components through an ordered series of events, where the decoding of each mRNA codon is accompanied by large-scale conformational changes within the ribosome and interacting factors, and between the ribosome and its mRNA and transfer RNA (tRNA) substrates. The need for large amounts of homogenous material has thwarted analogous investigations of the human translation mechanism. Hence, conserved and divergent features of the translation mechanism between single-cell organisms and mammals that determine the molecular basis of antibiotic specificity have remained largely obscure. Here, we seek to delineate common and distinct features of bacterial and human protein synthesis — and the translation mechanisms in healthy and cancerous human cells — to: 1] improve the efficacies of existing antibiotics; 2] develop new strategies for antibiotic interventions; and 3] explore the possibility of therapies targeting unchecked proliferative cell growth and metastatic spread. We will do so by establishing quantitative, structural and kinetic frameworks for the elemental steps of elongation in bacteria and humans using an integrated battery of biophysical methods, including single-molecule fluorescence imaging and state-of-the-art cryo-electron microscopy. Our collaborative investigations will delineate the order and timing of conformational events underpinning fidelity in bacterial and human elongation cycles and the structural and mechanistic distinctions that determine the efficacies of clinically relevant antibiotics targeting these processes. These insights will shed light on how translation control is achieved, reveal atomic-resolution descriptions drug action on bacterial and human ribosomes and inform opportunities for new interventions aimed at improving the efficacy and potency of clinical treatments for infectious pathogens and human disease.