Studies of Ribosome Biogenesis

核糖体生物发生的研究

• 批准号: 9902490
• 负责人: EDA KOCULI
• 金额: $ 34.39万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9902490
• 关键词: ATP phosphohydrolase; Acylation; Antibiotics; Bacteria; Binding Proteins; Biochemical; Biogenesis; C-terminal; Catalytic Domain; Cells; Chemicals; Crystallization; Data; Escherichia coli; Event; Family; Family member; Goals; High Pressure Liquid Chromatography; Hydroxyl Radical; In Vitro; Investigation; Knowledge; Laboratories; Life; Mass Spectrum Analysis; Modification; Molecular Chaperones; Molecular Structure; Molecular Weight; Nucleotides; Organism; Pathogenicity; Pathway interactions; Phenotype; Post-Transcriptional RNA Processing; Primer Extension; Process; Protein Biosynthesis; Proteins; Pseudouridine; Public Health; RNA; RNA Folding; RNA Helicase; RNA Processing; RNA Recognition Motif; Resolution; Ribosomal Proteins; Ribosomal RNA; Ribosomes; Role; Site; Specificity; Structural Protein; Structure; Sucrose; System; Time; World Health Organization; bacterial resistance; base; crosslink; design; experimental study; frontier; gel electrophoresis; helicase; in vivo; insight; interest; member; next generation sequencing; novel; particle; pathogenic bacteria; physical property; prevent; protein complex; structured data; three dimensional structure

The ribosome is the RNA-protein complex responsible for protein synthesis in every living cell. The Escherichia coli (E. coli) ribosome, which has a molecular weight of 2.5 MDa, consists of two subunits; the small (SSU) and the large subunit (LSU). The three-dimensional structure of the properly assembled LSU is known from crystallographic data; however, the knowledge of LSU assembly is limited. This knowledge is invaluable to facilitate the design of novel antibiotics targeting ribosome assembly. The goal of this proposal is to gain a detailed understanding of in vivo E. coli LSU assembly. The LSU assembly involves RNA folding, processing and modification, r-proteins binding, and the association and release of maturation factors. DbpA, a DEAD-box RNA helicase, is one of the LSU maturation factors. Expression of the helicase inactive DbpA construct, R331A, produces the accumulation of three LSU particles in-cell. The three particles convert over time to 50S LSU; hence, they are LSU assembly intermediates and not dead-end products of LSU assembly. Moreover, the three intermediates belong to three different stages of LSU assembly and three parallel pathways. Hence, their investigation will produce information on how different LSU assembly processes are coordinated and how different pathways of LSU assembly are interconnected. To our knowledge, this is the only system where three isolatable intermediates from three parallel pathways and different stages of LSU assembly accumulate in-cell; thus, this is the only system in which the coordination of assembly events and the interconnectivity of assembly pathways can be investigated. In Aim 1, the PI's laboratory will determine the rRNA structure, modification, processing, and the r-protein and maturation factor compositions of three LSU assembly particles. Comparing the rRNA structure, processing, modifications, and the r-protein and maturation factor compositions of the intermediates to each other and to the high-resolution crystal structure of the 50S subunit will identify: (i) rRNA structural isomerizations that must occur for LSU assembly; (ii) rRNA structural motifs' and r-proteins' role in rRNA post-transcriptional modification and processing; (iii) novel LSU maturation factors; (iv) common misfolded rRNA motifs in all LSU assembly pathways. The rRNA structure will be probed by chemical modification and Next-Generation Sequencing (NGS). The protein composition of the intermediates will be determined by mass spectrometry. In Aim 2, the PI's laboratory will investigate the rRNA regions DbpA catalytic core directly acts upon during LSU assembly in-cell and in the particles. UV cross-linking combined with NGS will be used to determine DbpA catalytic core regions of interaction with rRNA. The rRNA regions that the DbpA catalytic core directly contacts in-cell will inform on: (i) how many regions of the LSU DbpA acts upon, and (ii) how many pathways of LSU assembly involve DbpA. Determination of the DbpA catalytic core's native substrates in the particles combined with the knowledge of the misfolded rRNA structure from Aim 1 and the properly assembled 50S crystal structure data will determine DbpA's functional role on LSU assembly.

核糖体是负责每个活细胞中蛋白质合成的RNA蛋白质复合物。大肠疾病 大肠杆菌（大肠杆菌）核糖体的分子量为2.5 MDa，由两个亚基组成。小（SSU）和 大亚基（LSU）。从适当组装的LSU的三维结构是从中知道的 晶体学数据；但是，LSU组装的知识是有限的。这些知识对于 促进针对核糖体组装的新型抗生素的设计。该提议的目的是获得 详细了解体内大肠杆菌LSU组装。 LSU组件涉及RNA折叠，处理 和修饰，R蛋白结合以及成熟因子的关联和释放。 DBPA，一个死箱 RNA解旋酶是LSU成熟因子之一。解旋酶无活性DBPA构建体的表达， R331a在内部产生三个LSU颗粒的积累。这三个粒子随时间转换为50秒 lsu;因此，它们是LSU组装中间体，而不是LSU组装的死端产品。而且， 这三个中间体属于LSU组件的三个不同阶段和三个平行途径。因此， 他们的调查将提供有关不同LSU组装过程如何协调以及如何协调的信息 LSU组装的不同途径相互联系。据我们所知，这是唯一的系统 来自三个平行途径的可分离中间体和LSU组装的不同阶段积聚在内部； 因此，这是唯一的组装事件协调和组装互连的系统 可以研究途径。在AIM 1中，PI的实验室将确定RRNA结构，修改， 加工，以及三个LSU组装颗粒的R蛋白和成熟因子组成。比较 RRNA结构，加工，修饰以及R蛋白和成熟因子组成的组成 彼此之间的中间体和50S亚基的高分辨率晶体结构将识别：（i）rRNA LSU组装必须发生的结构异构化； （ii）rRNA结构基序和R蛋白在 RRNA转录后修改和处理； （iii）新型LSU成熟因子； （iv）常见 所有LSU组装途径中错误折叠的rRNA图案。 RRNA结构将通过化学探测 修改和下一代测序（NGS）。中间体的蛋白质组成将是 由质谱法确定。在AIM 2中，PI的实验室将调查rRNA区域DBPA 催化核直接在LSU组装内和颗粒中作用。紫外线交联 NGS将用于确定与RRNA相互作用的DBPA催化核心区域。 rRNA区域 DBPA催化核心直接接触核心将告知：（i）LSU DBPA ACT的数量有多少个区域 在和（ii）LSU组装的多少途径涉及DBPA。 DBPA催化核心的确定 颗粒中的天然底物与AIM 1和 正确组装的50S晶体结构数据将确定DBPA在LSU组装上的功能作用。