Studies of Ribosome Biogenesis

核糖体生物发生的研究

基本信息

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
项目类别：
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

项目摘要

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 RNA-蛋白质复合物，负责每个活细胞中的蛋白质合成。埃希氏菌属大肠杆菌（E. coli）核糖体，分子量为2.5 MDa，由两个亚基组成；小（SSU）和大亚基（LSU）。正确组装的 LSU 的三维结构可从晶体学数据；然而，LSU 组装的知识有限。这些知识对于促进针对核糖体组装的新型抗生素的设计。该提案的目标是获得详细了解体内大肠杆菌 LSU 组装。 LSU 组装涉及 RNA 折叠、加工和修饰、r 蛋白结合以及成熟因子的结合和释放。 DbpA，一个死盒 RNA解旋酶是LSU成熟因子之一。解旋酶失活 DbpA 构建体的表达， R331A，在细胞内产生三种 LSU 颗粒的积累。三个粒子随时间转换为50S 路易斯安那州立大学；因此，它们是 LSU 组装的中间体，而不是 LSU 组装的最终产品。而且，三个中间体属于 LSU 组装的三个不同阶段和三个平行途径。因此，他们的调查将产生有关如何协调不同路易斯安那州立大学组装流程以及如何协调的信息 LSU 组装的不同途径是相互关联的。据我们所知，这是唯一一个同时具备三个功能的系统来自三个平行途径和 LSU 组装不同阶段的可分离中间体在细胞内积累；因此，这是唯一一个能够协调集会活动和集会互连的系统可以研究途径。在目标 1 中，PI 实验室将确定 rRNA 结构、修饰、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中间体彼此之间以及 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转录后修饰和加工； (iii) 新的路易斯安那州立大学成熟因素； (四) 共同的所有 LSU 组装途径中错误折叠的 rRNA 基序。 rRNA结构将通过化学探测修饰和下一代测序（NGS）。中间体的蛋白质组成为通过质谱法测定。在目标 2 中，PI 实验室将研究 rRNA 区域 DbpA 在 LSU 组装过程中，催化核心直接作用于细胞内和颗粒中。 UV交联组合 NGS 将用于确定与 rRNA 相互作用的 DbpA 催化核心区域。 rRNA区域 DbpA 催化核心直接接触细胞内将告知：(i) LSU DbpA 有多少个区域发挥作用 (ii) 有多少 LSU 组装途径涉及 DbpA。 DbpA 催化核心的测定颗粒中的天然底物结合目标 1 中错误折叠的 rRNA 结构的知识和正确组装的 50S 晶体结构数据将确定 DbpA 在 LSU 组装中的功能作用。