Dynamics of Ribosomal Protein Synthesis

描述(由申请人提供)： 核糖体在细胞功能中的基本重要性被它在所有生命形式中的普遍性和基本保守性所证明。细菌核糖体的功能复合体在蛋白质合成过程中会发生较大的构象变化。最近在用X射线结晶学和低温电子显微镜阐明核糖体结构方面取得的巨大进展为这种变化提供了一些最好的证据。与此同时，对引发和延伸部分反应的详细速率研究，即使大部分是不完整的，也表明这些过程中的每一个都是复杂的多步骤反应，这提出了一个问题，即在多大程度上可以将特定的结构变化分配给拟议的动力学机制中描述的特定步骤。我们的工作总体目标是：a)使用荧光停止流、猝灭流和FRET方法充分阐明野生型核糖体的起始和延伸的动力学机制，重点放在三个G蛋白因子，EF-G，EF-Tu和IF2，以及它们都与之相互作用的核糖体位置，即所谓的GAC(GTP酶激活中心)；以及b)通过进行结构-功能分析，确定特定相互作用对特定核糖体催化步骤的重要性，测量蛋白质合成机制中的特定扰动如何影响特定蛋白质合成步骤的速率。TRNA的突变、肽转移酶中心(PTC)的突变以及针对GAC(硫代链球菌素)或PTC(恶唑烷酮和司帕霉素)的抗生素的加入都将引起特异性的干扰。我们工作将导致对核糖体功能的理解的提高，这可能对新的核糖体抗生素的设计、测试和使用产生重要影响，因为它应该提供一个确定特定抗生素对核糖体功能的特异性作用(S)的范式，这可以直接与抗生素结合位点的知识联系在一起。随着细菌对β-内酰胺类和喹啉类抗生素的耐药性变得越来越普遍，医学界对核糖体抗生素的兴趣也在增长。事实上，大型制药公司现在正投入大量资源，用于筛选和合理设计新的核糖体抗生素，以及修改已知的抗生素，以克服细菌耐药性，同时保持强大的抑制活性，这主要是由于新的恶唑烷酮类核糖体抗生素的成功开发和对细菌核糖体结构和功能的了解增加。

DESCRIPTION (provided by applicant): The fundamental importance of the ribosome in cellular function is evidenced by its universality and fundamental conservation throughout all forms of life. Functional complexes of the bacterial ribosome undergo large conformational changes during the course of protein synthesis. Recent dramatic progress in the elucidation of ribosome structure by both X-ray crystallography and cryoelectron microscopy has provided some of the best evidence for such changes. At the same time, detailed rate studies of initiation and elongation partial reactions, even though for the most part incomplete, have shown each of these processes to be complex, multistep reactions, raising the question of the extent to which specific structural changes can be assigned to specific steps described in proposed kinetic mechanisms. Our work has the overall goals of a) fully elucidating the kinetic mechanisms of initiation and elongation for wild-type ribosomes using fluorescence stopped-flow, quenched flow, and FRET approaches, focusing on three G- protein factors, EF-G, EF-Tu, and IF2, and the ribosomal site with which they all interact, the so-called GAC (GTPase Activation Center; and b) determining the importance of specific interactions for specific steps of ribosome catalysis by conducting structure-function analyses, measuring how specific perturbations in the protein synthesis machinery affect rates of specific steps of protein synthesis. The specific perturbations will be induced by mutation of tRNA, mutation of the peptidyl transferase center (PTC), and by addition of antibiotics targeting either the GAC (thiostrepton) or the PTC (oxazolidinones and sparsomycin). The improved understanding of ribosomal function that will result from our work could have important consequences for the design, testing, and utilization of new ribosomal antibiotics, since it should provide a paradigm for determining the specific effect(s) of a given antibiotic on ribosomal function, which can be linked directly to knowledge of the site of antibiotic binding. Medical interest in ribosomal antibiotics has been growing as bacterial resistance to beta-lactams and quinolines has become more widespread. Indeed, major pharmaceutical companies are now devoting considerable resources toward both the screening and rational design approaches for the discovery of new ribosomal antibiotics, as well as toward the modification of known antibiotics so as to overcome bacterial resistance while retaining strong inhibitory activity, largely as a result of the successful development of the new oxazolidinone family of ribosomal antibiotics and of the increased knowledge of the structure and function of the bacterial ribosome.