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 RNA 翻译成所有生物体中的蛋白质。过去十年在理解这种大蛋白质和核糖核酸结构的复杂结构方面取得了显着进展。尽管在确定核糖体结构方面取得了重大进展，但有关该复合物生物发生的知识仍然存在许多重要的空白。该项目将研究成熟真核小亚基的直接前体的结构，即 40S 前晚期组装中间体。拟议的工作将应用电子冷冻显微镜（cryo-EM）单粒子分析来纯化前 40S 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了解核糖体成熟的最终序列所需的重要信息。 40 年代末晚期向成熟、有功能的核糖体转变的一个重要步骤是 D 位点内转录间隔区 1 (ITS-1) 的裂解。使用冷冻电镜图谱，人们将能够追踪rRNA 通过负责其切割的核酸内切酶组装因子 Nob1 的路径。此外，该图谱将显示与该裂解过程的调节相关的蛋白质如何与该位点在结构上相互作用。此外，分子动力学灵活拟合将用于将已知的成熟真核生物 40S 亚基晶体结构建模为获得的前 40S 冷冻电镜图。这将允许绘制成熟亚基及其前体中核糖体蛋白和 rRNA 二级结构的位置之间的差异，从而使我们能够获得最终成熟过程的重要机制见解。 公共健康相关性：关键细胞机器核糖体的构建错误可能导致多种疾病，包括罕见的贫血症。拟议的工作将研究核糖体发育过程中重要中间步骤的结构。希望通过绘制该结构，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理解相关病理学和设计新治疗策略的结构框架。