Dynamics of Ribosomal Protein Synthesis

描述（由申请人提供）： 核糖体在细胞功能中的根本重要性由其在所有生命形式中的普遍性和基本保守性证明。 细菌核糖体的功能复合物在蛋白质合成过程中经历大的构象变化。 最近的戏剧性的进展，阐明核糖体结构的X射线晶体学和冷冻电子显微镜提供了一些最好的证据，这种变化。 与此同时，详细的速率研究的起始和延伸部分反应，即使在大多数情况下不完整，这些过程中的每一个是复杂的，多步骤的反应，提出了问题的程度，具体的结构变化可以分配到特定的步骤中所描述的建议的动力学机制。 我们的工作的总体目标是：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起始和延伸的动力学机制，重点是三个G蛋白因子EF-G、EF-Tu和IF 2，以及它们都相互作用的核糖体位点，即所谓的GAC（GTSTAVE激活中心;和B）通过进行结构-功能分析确定特定相互作用对于核糖体催化的特定步骤的重要性，测量蛋白质合成机制中的特定扰动如何影响蛋白质合成的特定步骤的速率。 通过tRNA突变、肽基转移酶中心（PTC）突变以及通过添加靶向GAC（硫链丝菌素）或PTC（恶唑烷酮和稀疏霉素）的抗生素来诱导特异性扰动。 从我们的工作中得到的对核糖体功能的更好理解可能对新的核糖体抗生素的设计，测试和利用产生重要影响，因为它应该提供一个范例来确定给定抗生素对核糖体功能的具体影响，这可以直接与抗生素结合位点的知识联系起来。 随着细菌对β-内酰胺类和喹啉类的耐药性变得更加普遍，对核糖体抗生素的医学兴趣也在增长。事实上，主要的制药公司现在正投入大量资源用于发现新的核糖体抗生素的筛选和合理设计方法，以及对已知抗生素的修饰，以克服细菌耐药性，同时保持强抑制活性，这主要是由于核糖体抗生素的新恶唑烷酮家族的成功开发以及对细菌核糖体的结构和功能。

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.