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Studies of Ribosome Biogenesis

核糖体生物发生的研究

基本信息

批准号：
10598540
负责人：
EDA KOCULI
金额：
$ 32.24万
依托单位：
UNIVERSITY OF TEXAS EL PASO
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 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10598540
关键词：
ATP phosphohydrolase Acylation Antibiotics Bacteria Binding Proteins Biochemical Biogenesis C-terminal Catalytic Domain Cells Chemicals Crystallography Data Escherichia coli Event Family Family member Goals High Pressure Liquid Chromatography Humanities Hydroxyl Radical In Vitro Investigation Isomerism 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 Structure Sucrose System Time World Health Organization bacterial resistance base crosslink design experimental study frontier gel electrophoresis helicase in vivo insight interest member migration next generation sequencing novel particle pathogenic bacteria physical property prevent protein complex 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，在每个活细胞中负责蛋白质合成。的大肠