Atomic-Resolution Analysis of Translation Control

描述（由申请人提供）：核糖体在细胞中直接将基因型与表型偶联，其调控是细胞生理学的核心。尽管自最后一个共同祖先以来，翻译的许多步骤已经分道扬镳，但在细菌和人类中，翻译起始和核糖体再循环仍然密切相关。翻译周期中的这些耦合事件是本应用的重点。在过去的十年中，我们对蛋白质合成的分子机制的理解经历了一场革命，这是建立在核糖体结构生物学的快速发展之上的。高分辨率结构现在可以用于细菌和真核生物的整个核糖体，以及古生菌的大核糖体亚基。然而，由于分离结构中间体的挑战，与翻译中动态事件有关的重要问题仍未得到解答。在这个应用中，我们提出探索细菌核糖体循环的分子机制，这是一个由GTPase延伸因子G催化的过程，也是抗生素的有效靶点。我们将结合x射线晶体学、低温电子显微镜（cryo-EM）和单分子生物物理学，在这一领域取得突破性成果。通过使用x射线晶体学，冷冻电镜，以及我们开发的研究人类翻译起始因子eIF3的新方法和工具，我们也将探索人类翻译起始，蛋白质合成中最具活力的步骤之一。我们将利用我们独特的系统功能重构人类翻译起始因子eIF3，冷冻电镜，粗神经孢子虫遗传学，以及基因组工程中革命性的新方法来剖析eIF3对人类细胞mrna启动密码子选择的贡献。结合大肠杆菌中的eIF3重组，N. crassa中的遗传学，人类细胞中的诱变以及尖端的冷冻电镜技术，为揭示eIF3在人类翻译起始中的分子贡献提供了一种强大而独特的手段。综上所述,

DESCRIPTION (provided by applicant): Protein synthesis by the ribosome directly couples genotype to phenotype in the cell, and its regulation is central to cellular physiology. Although many steps of translation have diverged since the last common ancestor, translation initiation and ribosome recycling remain intimately coupled in both bacteria and humans. These coupled events in the translation cycle are the focus of the present application. Our understanding of the molecular mechanism of protein synthesis has undergone a revolution in the last decade, built on rapid advances in the structural biology of the ribosome. High-resolution structures are now available for the entire ribosome in both bacteria and eukaryotes, and of the large ribosomal subunit in archaea. However, important questions relating to dynamic events in translation remain unanswered due to the challenge of isolating structural intermediates. In this application, we propose to probe the molecular mechanism of ribosome recycling in bacteria, a process catalyzed by the GTPase elongation factor G and a validated target of antibiotics. We will build on our prior groundbreaking results in this area by combining x-ray crystallography, cryo-electron microscopy (cryo-EM) and single-molecule biophysics. By using x-ray crystallography, cryo-EM, and new methods and tools we have developed to study human translation initiation factor eIF3, we will also probe human translation initiation, one of the most dynamic steps in protein synthesis. We will use our unique system to functionally reconstitute human translation initiation factor eIF3, cryo-EM, genetics in Neurospora crassa, and revolutionary new methods in genome engineering to dissect the contribution of eIF3 to start codon selection on human cellular mRNAs. The combination of eIF3 reconstitution in E. coli, genetics in N. crassa, mutagenesis in human cells, and cutting-edge cryo-EM provides a powerful and unique means to unravel the molecular contributions of eIF3 to translation initiation in humans. Taken together, the three aims of this application build on the fundamental insights into ribosome structure and function obtained in the prior funding period, and address key mechanisms in translational control that could be exploited for the development of new antibiotics and therapeutics.