Imaging Protein Synthesis on the Ribosome using Single-Molecule FRET

项目摘要： 细胞内蛋白质合成及其调控机制决定了细胞内蛋白质合成的多样性和能力， 蛋白质组这种调节控制的中心整合点是核糖体：一个两个亚基，兆道尔顿 RNA-蛋白质组装。突出了翻译和核糖体对调节的敏感性， 大多数已知的抗生素要么失调要么阻断核糖体功能。相应地， 蛋白质合成机制的分子细节有可能告知基因表达的范例 我们将继续就如何控制和应对新出现的耐药病原体对全球健康的威胁提供咨询意见。为损失 翻译控制是癌症的标志，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了靶向治疗策略的希望。 对制约翻译机制的结构-功能关系的研究主要是 在细菌中使用传统的集成方法进行。这些研究表明， 蛋白质从信使RNA（mRNA）合成的翻译，称为延伸，是最长的时间 密集和通常药物靶向。他们还发现，延伸需要核糖体瞬时 通过一系列有序的事件与特定的细胞成分相互作用，其中每个细胞成分的解码 mRNA密码子伴随着核糖体和相互作用因子内的大规模构象变化， 以及核糖体与其mRNA和转运RNA（tRNA）底物之间的相互作用。需要大量的 同质材料阻碍了对人类翻译机制的类似研究。因此，我们认为， 单细胞生物和哺乳动物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的研究仍然很模糊。在此，我们寻求 描述了细菌和人类蛋白质合成的共同和独特特征-以及翻译 在健康和癌症人类细胞中的机制-以：1]提高现有抗生素的功效; 2] 开发抗生素干预的新策略; 3]探索针对未经检查的治疗的可能性 增殖细胞生长和转移扩散。我们将通过建立定量、结构和动力学模型来实现这一目标。 框架的基本步骤的延长在细菌和人类使用一个集成的电池， 生物物理方法，包括单分子荧光成像和最先进的冷冻电子 显微镜我们的合作调查将描绘构象事件的顺序和时间 在细菌和人类的延伸周期和结构和机制的区别的基础保真度 确定针对这些过程的临床相关抗生素的功效。这些见解将使 如何实现翻译控制的光，揭示原子分辨率的描述药物对细菌的作用， 人类核糖体和信息的机会，新的干预措施，旨在提高疗效和效力， 临床治疗传染性病原体和人类疾病。

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.