Structural Requirements for Ribosome-Dependent GTPase Activation and Hybrid State

总结 ! 在蛋白质合成过程中，转运RNA（tRNAs）和信使RNA（mRNA）以一种 通过核糖体的协调方式，一个过程被称为“核糖体易位”。As易位 发生时，形成许多瞬时中间体，由此核糖体结合的tRNA底物采用几种 不同的构象。同时，70 S核糖体通过不同的构象旋转， 在翻译的延伸阶段调节tRNA的移动。这个过程是严格的 受核糖体依赖性GTP酶延伸因子G（EF-G）的调节，EF-G瞬时结合到 核糖体亚基界面，其中GTP被水解以允许进行快速和准确的翻译。 在这项研究中，化学足迹和亚基间FRET程序将被用来研究 EF-G的结构要求，用于稳定核糖体易位的第一步， 混合型国家。其次，一种新描述的核糖体转位酶， 命名为延伸因子4（EF 4），不同易位状态下的70 S核糖体将进一步被 介绍了第三，核糖体蛋白L7/L12在核糖体依赖性GT3结合中的作用， 通过70 S核糖体的激活将通过GTdR活性测定和用于GTdR的方法来定义。 EF-G平衡结合的定量测量。最后，几种溶液的动态结构状态 在不同鸟嘌呤核苷酸存在下的核糖体依赖性GTP酶的特征在于： 小角X射线散射（SAXS）方法研究生物大分子。 在这项研究中，我们建议首次调查EF-G的域要求， 稳定的杂交状态和EF 4催化的反向易位的结构机制， 溶液此外，我们假设，高度纯化的核糖体制备物消耗了 核糖体蛋白L7/L12，以及用于重建的L7/L12的重组形式，将导致 明确描述了其在GT3结合和催化中的作用。最后，我们将揭开动态的神秘面纱 核糖体依赖性GTP酶在核糖体功能复合物识别方面的性质。 这项拟议的工作将利用PI在足迹，SAXS和核糖体生物化学方面的专业知识。 公共卫生相关性：用于临床治疗细菌感染的最有效抗生素 感染靶向核糖体。因此，更好地了解核糖体的结构和功能 将有助于发现新的药物，解决日益严重的耐药致病菌问题。 此外，发生在核糖体内的阻止正常功能的突变与 遗传性疾病，如耳聋和癌症。 !

Summary ! During protein synthesis, transfer RNAs (tRNAs) and messenger RNA (mRNA) move in a coordinated manner through the ribosome, a process denoted as 'ribosomal translocation'. As translocation occurs, many transient intermediates are formed whereby ribosome-bound tRNA substrates adopt several different conformations. Concomitantly, the 70S ribosome rotates through different conformers to accommodate the movement of tRNA during the elongation phase of translation. This process is strictly regulated by the ribosome-dependent GTPase elongation factor G (EF-G), which binds transiently to the ribosome subunit interface where GTP is hydrolyzed to allow for rapid and accurate translation to proceed. In this study, chemical footprinting and intersubunit FRET procedures will be employed to study the structural requirements of EF-G for stabilization of the first step of ribosomal translocation, the formation of the hybrid state. Secondly, the nature of the interaction between a newly described ribosomal translocase, termed elongation factor 4 (EF4), and the 70S ribosome under different translocation states will be further described. Third, the role of ribosomal protein L7/L12 in ribosome-dependent GTPase binding and activation by the 70S ribosome will be defined through GTPase activity assays and methods for the quantitative measure of EF-G equilibrium binding. Lastly, the dynamic, solution structure states of several ribosome-dependent GTPases in the presence of different guanine nucleotides will be characterized with small angle x-ray scattering (SAXS) approaches to study biological macromolecules. In this study, we propose to investigate for the first time the domain requirements of EF-G for stabilization of the hybrid state and the structural mechanism of EF4-catalyzed reverse translocation in solution. Furthermore, we hypothesize that the highly purified preparations of ribosomes depleted in ribosomal protein L7/L12, as well as a recombinant form of L7/L12 for reconstitution, will lead to an unambiguous description of its role in GTPase binding and catalysis. Lastly, we will demystify the dynamic nature of ribosome-dependent GTPases in regards to the recognition of ribosome functional complexes. This proposed work will capitalize on the PI's expertise in footprinting, SAXS, and ribosome biochemistry. Public Health Relevance: The most effective antibiotics used for clinical treatments of bacterial infections target ribosomes. Therefore, a better understanding of the structure and function of the ribosome will aid in finding new drugs that address the increasing problem of antibiotic-resistant pathogenic bacteria. Furthermore, mutations that occur within ribosomes that prevent proper function have been associated with genetic diseases, such as deafness and cancer. !