Dynamics of Ribosomal Protein Synthesis

描述（由申请人提供）：核糖体通过翻译循环催化蛋白质合成。对细菌核糖体结构的研究提供了有力的证据，证明细菌核糖体的功能复合物在此循环过程中经历了大的构象变化。与此同时，对翻译循环的部分反应的研究表明，这些过程是复杂的、多步骤的反应，我们对这些反应的了解仍然很不完整。我们的一般方法，涉及到几个平行测量的结果相结合，以允许制定定量动力学方案描述这样的部分反应。这样的定量计划允许阐明的时间内的构象变化的过程中，和精确的机制，重要的变量影响核糖体催化功能。将翻译循环分解成其组成部分允许确定给定抗生素对核糖体功能的特定作用。这些知识与已知的抗生素结合位点有关，对于设计和评估新的抗生素是有用的。我们的具体目标是：1.荧光试剂的制备与应用。我们将制备荧光核糖体配体和标记在特定蛋白质中的荧光核糖体。这些荧光试剂将用于目标2-5中的快速动力学实验。2.延长周期。我们将确定循环中的特定步骤如何受到以下因素的影响：a）E位点占据，B）参与肽键形成的tRNA的身份，c）与核糖体结合的新生肽链的大小和序列，d）tRNA和核糖体RNA的突变，以及e）添加的抗生素。我们也将使用冷冻电子显微镜（cryoEM）来阐明易位中间体的结构，并制定一个定量的机制，LepA催化的回易位。3.入会仪式。起始的关键步骤是形成70 S起始复合物（70 SIC）。我们将i）确定70 SIC形成的动力学方案如何受到起始密码子变化的影响; ii）阐明70 SIC形成过程中关键构象变化的时间和幅度; iii）将噻唑类抗生素对70 SIC形成的影响与已经发现的硫链丝菌素的影响进行比较，作为开发新疗法过程的一部分。4. G因子蛋白与核糖体的同时结合。我们将采用cryoEM和建模来获得当两个G蛋白IF 2和EF-Tu在从起始到延伸的过渡期间同时与核糖体结合时形成的复合物的结构，并将这些研究与动态测量相结合，这些动态测量旨在捕获IF 2的运动作为EF-Tu结合。我们还将确定依赖于复合物的形成上的核糖体蛋白质二聚体L7/L12的化学计量。将对EF-G和EF-Tu进行类似的研究，以测试它们是否可以同时与核糖体结合，以在延伸过程中形成短暂的中间体。5.单分子FRET实验将用于鉴定作为目标2和4的一部分研究的反应途径中的瞬时中间体。核糖体通过翻译循环催化蛋白质合成，其在细胞功能中的根本重要性通过其在所有生命形式中的普遍性和根本保守性得到证明。我们提出的研究将有助于进一步阐明核糖体的动态作用机制，并为确定给定抗生素对核糖体功能的特定作用提供范例。这些知识在设计和评价由于目前临床使用的抗生素的耐药性的广泛发展而迫切需要的新抗生素方面具有明确的效用。

DESCRIPTION (provided by applicant): Ribosomes catalyze protein synthesis via the translation cycle. Studies of bacterial ribosome structure provide strong evidence that functional complexes of the bacterial ribosome undergo large conformational changes during the course of this cycle. At the same time, studies of partial reactions of the translation cycle have shown these processes to be complex, multistep reactions, about which our knowledge remains quite incomplete. Our general approach, involves combining the results of several measurements made in parallel to allow formulation of quantitative kinetic schemes describing such partial reactions. Such quantitative schemes allow elucidation of the timing of conformational changes within a process, and the precise mechanisms by which important variables affect ribosome catalytic function. Resolving the translation cycle into its component parts permits determination of the specific effect(s) of a given antibiotic on ribosomal function. Such knowledge, linked to known sites of antibiotic binding, is useful for the design and evaluation of new antibiotics. Our specific aims are: 1. Preparation and utilization of fluorescent reagents. We will prepare fluorescent ribosomal ligands and fluorescent ribosomes labeled in specific proteins. These fluorescent reagents will be used in rapid kinetics experiments in Aims 2-5. 2. The elongation cycle. We will determine how specific steps within the cycle are affected by a) E-site occupation, b) the identities of the tRNAs participating in peptide bond formation, c) the size and sequence of the nascent peptide chain bound to the ribosome, d) mutations in tRNA and ribosomal RNA, and e) added antibiotics. We also will use cryoelectronmicroscopy (cryoEM) to elucidate the structures of intermediates in translocation and formulate a quantitative mechanism for LepA catalysis of back translocation. 3. Initiation. A key step in initiation is the formation of a 70S initiation complex (70SIC). We will i) determine how the kinetic scheme for 70SIC formation is affected by changes in the initiation codon; ii) elucidate the timing and magnitude of a key conformational change within the process of 70SIC formation; and iii) compare the effects of thiazole antibiotics on 70SIC formation with those already found for thiostrepton, as part of the process of developing new therapeutics. 4. Simultaneous binding of G-factor proteins to the ribosome. We will employ cryoEM and modeling to obtain a structure of the complex that is formed when two G-proteins, IF2 and EF-Tu bind to the ribosome simultaneously during the transition from initiation to elongation, and couple such studies with dynamic measurements designed to both capture the movement of IF2 as EF-Tu binds. We also will determine the dependence of complex formation on the stoichiometry of ribosomal protein dimer L7/L12. Similar studies will be conducted on EF-G and EF-Tu to test whether they might bind simultaneously to the ribosome to form a short-lived intermediate during elongation. 5. Single molecule FRET experiments will be used to identify transient intermediates in reaction pathways studied as parts of Aims 2 and 4. PUBLIC HEALTH RELEVANCE The ribosome catalyzes protein synthesis via the translation cycle, and its fundamental importance in cellular function is evidenced by its universality and fundamental conservation throughout all forms of life. The studies we propose will contribute importantly to the further elucidation of the dynamic mechanism of action of the ribosome, and provide a paradigm for determining the specific effect(s) of a given antibiotic on ribosomal function. Such knowledge is of clear utility in the design and evaluation of new antibiotics that are urgently needed due to the widespread development of resistance to antibiotics currently in clinical use.