Structural Studies of a Ribosomal 40S Subunit Biogenesis Intermediate

描述(申请人提供)：核糖体负责在所有活着的生物体中将RNA翻译成蛋白质。在过去的十年里，在理解这种大型蛋白质和核糖核酸结构的复杂结构方面取得了显着的进展。尽管在确定核糖体结构方面取得了重大进展，但关于这种复合体的生物发生的知识仍然存在许多重要的空白。这个项目将研究成熟的、真核的、小亚基的直接前体的结构，40年代末的前组装中间体。拟议的工作将应用电子冷冻显微镜(Cryo-EM)单颗粒分析纯化的Pre-40s亚基，以获得亚纳米分辨率的MAP。这种高分辨率冷冻-EM结构的实现将使人们能够获得理解核糖体成熟的最终序列所必需的重要信息。在40年代末向成熟、有功能的核糖体过渡的过程中，重要的一步是D位的内部转录间隔区1(ITS-1)的切割。使用冷冻-EM图，人们将能够通过负责切割rRNA的核酸内切酶组装因子Nob1追踪rRNA的路径。此外，这张地图将显示与这一分裂过程的调节相关的蛋白质如何在结构上与这个位点相互作用。此外，分子动力学柔性拟合将被用来将已知的成熟真核生物40S亚单位晶体结构模拟到获得的前40S的冷冻EM图中。这将使我们能够定位成熟亚基及其前体中核糖体蛋白和rRNA二级结构的位置之间的差异，从而使我们能够从机制上获得对最终成熟过程的关键见解。 与公共卫生相关：核糖体这一关键的细胞机器的结构错误可导致多种疾病，包括罕见形式的贫血。这项拟议的工作将研究核糖体发育的重要中间步骤的结构。希望通过绘制这一结构图，更好地了解核糖体发育的关键步骤。这些知识将使我们能够构建理解相关病理和设计新的治疗策略的结构框架。

DESCRIPTION (provided by applicant): Ribosomes are responsible for translating RNA into proteins in all living organisms. The last ten years have shown remarkable advances in understanding the complex architecture of this large protein and ribonucleic acid structure. Despite the significant progress in determining the ribosome structure, knowledge on the biogenesis of this complex still contains many important gaps. This project will study the structure of the immediate precursor to the mature, eukaryotic, small subunit, the late pre-40S assembly intermediate. The proposed work will apply electron cryomicroscopy (cryo-EM) single particle analysis to purified pre-40S subunits to obtain a map at sub-nanometer resolution. Achievement of this high-resolution cryo-EM structure will allow access to important information necessary to understand the final sequence of ribosome maturation. An important step in the transition of the late pre-40S to a mature, functioning ribosome is the cleavage of the internally transcribed spacer 1 (ITS-1) at site D. Using the cryo-EM map, one will be able to trace the path of the rRNA through the endonuclease assembly factor responsible for its cleavage, Nob1. Furthermore, this map will show how proteins associated with regulation of this cleave process interact structurally with this site. Additionally, molecular dynamics flexible fitting will be use to model the known mature eukaryotic 40S subunit crystal structure into the obtained cryo-EM map of the pre-40S. This will allow mapping of differences between the locations of ribosomal proteins and rRNA secondary structure in the mature subunit and its precursor, thereby allowing us to obtain crucial mechanistic insights in to the final maturation process. PUBLIC HEALTH RELEVANCE: Errors in the construction of a key cellular machine, the ribosome, can cause a number of diseases, including rare forms of anemia. The proposed work will study the structure of an important intermediate step in the development of the ribosome. It is hoped that through mapping this structure, key steps of ribosome development will be better understood. This knowledge will allow us to construct the structural framework for understanding the related pathologies and for designing novel therapeutic strategies.