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:
- 金额:$ 32.24万
- 依托单位:
- 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:
- 关键词:ATP phosphohydrolaseAcylationAntibioticsBacteriaBinding ProteinsBiochemicalBiogenesisC-terminalCatalytic DomainCellsChemicalsCrystallographyDataEscherichia coliEventFamilyFamily memberGoalsHigh Pressure Liquid ChromatographyHumanitiesHydroxyl RadicalIn VitroInvestigationIsomerismKnowledgeLaboratoriesLifeMass Spectrum AnalysisModificationMolecular ChaperonesMolecular StructureMolecular WeightNucleotidesOrganismPathogenicityPathway interactionsPhenotypePost-Transcriptional RNA ProcessingPrimer ExtensionProcessProtein BiosynthesisProteinsPseudouridinePublic HealthRNARNA FoldingRNA HelicaseRNA ProcessingRNA Recognition MotifResolutionRibosomal ProteinsRibosomal RNARibosomesRoleSiteSpecificityStructureSucroseSystemTimeWorld Health Organizationbacterial resistancebasecrosslinkdesignexperimental studyfrontiergel electrophoresishelicasein vivoinsightinterestmembermigrationnext generation sequencingnovelparticlepathogenic bacteriaphysical propertypreventprotein complexthree 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
项目成果
期刊论文数量(2)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
RNA Post-Transcriptional Modifications in Two Large Subunit Intermediates Populated in E. coli Cells Expressing Helicase Inactive R331A DbpA.
- DOI:10.1021/acs.biochem.2c00096
- 发表时间:2022-05-17
- 期刊:
- 影响因子:2.9
- 作者:Koculi, Eda;Cho, Samuel S.
- 通讯作者:Cho, Samuel S.
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EDA KOCULI其他文献
EDA KOCULI的其他文献
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