Structural Studies of a Ribosomal 40S Subunit Biogenesis Intermediate

描述（由申请人提供）：核糖体负责将RNA翻译成所有生物体中的蛋白质。近十年来，在了解这种大型蛋白质的复杂结构和核糖核酸结构方面取得了显著进展。尽管在确定核糖体结构方面取得了重大进展，但对该复合体的生物发生的了解仍然存在许多重要的空白。该项目将研究成熟的真核小亚基，40s前组装中间体的直接前体结构。这项工作将应用电子低温显微镜（cryo-EM）单粒子分析纯化40s前亚基，以获得亚纳米分辨率的图谱。这种高分辨率冷冻电镜结构的实现将允许获得必要的重要信息，以了解核糖体成熟的最终序列。在前期40s晚期向成熟的功能核糖体过渡的一个重要步骤是内部转录间隔1 （its -1）在d位点的切割。使用低温电镜图谱，人们将能够通过负责切割的内切酶组装因子Nob1来追踪rRNA的路径。此外，该图谱将显示与该切割过程调控相关的蛋白质如何在结构上与该位点相互作用。此外，分子动力学柔性拟合将用于将已知成熟的真核40S亚基晶体结构建模到获得的40S前的cryo-EM图谱中。这将允许绘制成熟亚基及其前体中核糖体蛋白和rRNA二级结构位置之间的差异，从而使我们能够在最终成熟过程中获得关键的机制见解。

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.