Atomic-Resolution Analysis of Translation Control

描述（由申请人提供）：核糖体的蛋白质合成直接将细胞中的基因型与表型偶联，并且其调节是细胞生理学的中心。尽管自最后一个共同祖先以来，翻译的许多步骤已经分化，但翻译起始和核糖体再循环在细菌和人类中仍然密切相关。翻译周期中的这些耦合事件是本申请的焦点。我们对蛋白质合成的分子机制的理解在过去十年中经历了一场革命，建立在核糖体结构生物学的快速发展之上。高分辨率的结构，现在可用于整个核糖体在细菌和真核生物，和大亚基在古细菌的核糖体。然而，由于分离结构中间体的挑战，与翻译中的动态事件相关的重要问题仍然没有答案。在这个应用中，我们提出了探测细菌中核糖体再循环的分子机制，这是一个由GT延伸因子G催化的过程，也是一个经过验证的抗生素靶点。我们将建立在我们之前在这一领域的突破性成果，结合X射线晶体学，冷冻电子显微镜（cryo-EM）和单分子生物物理学。通过使用X射线晶体学，cryo-EM，以及我们开发的研究人类翻译起始因子eIF 3的新方法和工具，我们还将探索人类翻译起始，这是蛋白质合成中最具活力的步骤之一。我们将使用我们独特的系统来功能性地重建人类翻译起始因子eIF 3，cryo-EM，粗糙脉孢菌的遗传学，以及基因组工程中革命性的新方法来剖析eIF 3对人类细胞mRNA起始密码子选择的贡献。eIF 3在E. coli，N. crassa、人类细胞诱变和尖端cryo-EM为揭示eIF 3对人类翻译起始的分子贡献提供了强大而独特的手段。综合起来看， 该申请的三个目标建立在对核糖体结构和功能的基本认识的基础上，并解决了翻译控制中的关键机制，这些机制可用于开发新的抗生素和治疗方法。

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.