Structural Studies of a Ribosomal 40S Subunit Biogenesis Intermediate

核糖体 40S 亚基生物发生中间体的结构研究

• 批准号: 8393777
• 负责人: Jeffrey Franklin Herbstman
• 金额: $ 1.77万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8393777
• 关键词: Achievement; Anemia; Architecture; Back; Behavior; Binding; Biochemical; Biogenesis; Classification; Cleaved cell; Collection; Complex; Cryoelectron Microscopy; Data; Data Collection; Data Set; Development; Diamond-Blackfan anemia; Disease; Docking; Dyskeratosis Congenita; Eukaryota; Exhibits; Future; Knowledge; Life; Location; Maps; Messenger RNA; Methods; Modeling; Organism; Pathology; Physics; Play; Positioning Attribute; Process; Protein Biosynthesis; Proteins; RNA; Regulation; Resolution; Ribosomal Proteins; Ribosomal RNA; Ribosomes; Role; Shapes; Site; Sorting - Cell Movement; Staging; Structure; Techniques; Translating; Translation Initiation; Work; design; endonuclease; flexibility; insight; molecular dynamics; nanometer; novel therapeutics; particle; premature; prevent; programs; reconstruction; research study; statistics

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.

描述（由申请人提供）：核糖体负责将所有生物体中的RNA翻译成蛋白质。在过去的十年里，人们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，但关于这种复合物的生物起源的知识仍然存在许多重要的空白。本项目将研究直接前体结构到成熟的真核小亚基、后期前40 S组装中间体。拟议的工作将应用电子低温显微镜（cryo-EM）单粒子分析纯化前40 S亚基，以获得亚纳米分辨率的地图。实现这种高分辨率的cryo-EM结构将允许访问重要的信息，了解核糖体成熟的最终序列。 在晚期前40 S向成熟的功能性核糖体转变的过程中，一个重要步骤是D位点的内转录间隔区1（ITS-1）的切割。使用cryo-EM图谱，将能够追踪rRNA通过负责其切割的核酸内切酶组装因子Nob 1的路径。此外，该图谱将显示与该切割过程的调节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的成熟真核40 S亚基晶体结构建模到所获得的前40 S的冷冻-EM图中。这将允许映射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，从而使我们能够获得最终成熟过程中的关键机制见解。 公共卫生相关性：核糖体是一个关键的细胞机器，它的构造错误会导致许多疾病，包括罕见的贫血症。拟议的工作将研究核糖体发育过程中一个重要中间步骤的结构。希望通过绘制这种结构，可以更好地理解核糖体发育的关键步骤。这些知识将使我们能够构建结构框架，以了解相关的病理和设计新的治疗策略。