The Role of Zinc ions for RNA Binding and Catalytic Function of the DYW-deaminase
锌离子对 RNA 结合的作用和 DYW 脱氨酶的催化功能
基本信息
- 批准号:9765338
- 负责人:
- 金额:$ 14.6万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2017
- 资助国家:美国
- 起止时间:2017-09-12 至 2021-08-31
- 项目状态:已结题
- 来源:
- 关键词:Amino Acid SubstitutionAmino AcidsAntibody DiversityBindingBinding SitesBiological AssayC-terminalCatalysisCell physiologyChloroplastsCrystallizationCrystallographyCytidineCytidine DeaminaseDNADataDeaminaseDeaminationEdetic AcidEndoribonucleasesEnzymesEventFutureGlutamatesGoalsHydrolaseHydroxyl RadicalIn VitroIonsLigand BindingLigandsLinkLocationMeasuresMetal Binding SiteMetalsMitochondriaModelingMutationNucleic Acid BindingNucleic AcidsNucleotide DeaminasesNucleotidesPlastidsProteinsProtonsPyrimidineRNARNA BindingRNA EditingReactionRecombinant ProteinsRecombinantsRibonucleasesRibonucleotidesRoleSeriesSiteSpecificityStructureSurveysSystemTestingTranscriptVascular PlantViralZincanalogexperimental studyin vitro activityinnovationmetal chelatornew therapeutic targetnovelstoichiometry
项目摘要
Project Summary/Abstract
RNA editing and cleavage of chloroplast transcripts require proteins with the DYW-deaminase domain. The
DYW-deaminase shares several features common to nucleotide deaminases and comprises the enzymatic
portion of the RNA editing machinery. Some DYW-deaminase proteins are not associated with any RNA
editing function and are required for ribonuclease cleavage. On this basis the domain can be broken down into
one class with RNA editing functions and a second class with ribonuclease functions. Related enzymes
function exclusively as amino hydrolases and are critical for various cellular functions including RNA editing,
antibody diversity, and viral defense. Each DYW-deaminase domains binds two zinc ions. Zinc #1 is most likely
coordinated by the typical C/HAE---CXXC motif found in all nucleotide deaminases. Amino acids in a unique C-
terminus likely coordinate Zinc #2 and are nearby the catalytic zinc ion binding site. A general mechanism for
cytidine deamination does not require two zinc ions. The second zinc ion could have a unique co-catalytic
function, serve as a structural feature, or have a role in ligand binding. Zinc ions are not required for all
ribonuclease mechanisms and addition of EDTA promotes the ribonuclease cleavage activity of recombinant
DYW-deaminases in vitro. One or both zinc ions might operate as a zinc “switch” that governs the deaminase
and ribonuclease functions of the domain.
This project aims to investigate a model where zinc ions act as a “switch” that determines catalytic
function. Zinc binding in DYW-deaminases with endoribonuclease and RNA editing functions will be compared
to investigate if amino acid differences present in each functional classes determine zinc stoichiometry. A pool
of recombinant DYW-deaminases with amino acid substitutions in putative zinc coordinating residues will be
created to pinpoint the location of zinc coordinating residues. Endoribonuclease activities will be assayed for
rDYW-deaminase proteins with known RNA editing functions as well as known endoribonuclease functions in
the presence or absence of metal chelators. Ribonuclease activity will be measured for recombinant proteins
with altered zinc stoichiometries and mutations in zinc coordinating residues. A series of proteins with amino
acid changes might discover a link between zinc binding and enzymatic function as a well as to establish the
reaction mechanism for RNA cleavage. An activity assay for RNA editing that includes addition of exogenous
DYW-deaminases will be developed. The activity of DYW-deaminases with different zinc stiochiometries will be
measured to determine the relationship between zinc ion binding and aminohydrolase activity. The second zinc
ion may have a specialized role in ligand binding and to test this possibility the nucleic acid ligand requirements
of the DYW-deaminase will be determined using RNAs and putative substrates with modified nucleotides.
Finally, crystallization conditions for recombinant proteins will be screened. Crystals could be used in future
experiments aimed at solving the structure of the DYW-deaminase.
Project Summary/Abstract
RNA editing and cleavage of chloroplast transcripts require proteins with the DYW-deaminase domain. The
DYW-deaminase shares several features common to nucleotide deaminases and comprises the enzymatic
portion of the RNA editing machinery. Some DYW-deaminase proteins are not associated with any RNA
editing function and are required for ribonuclease cleavage. On this basis the domain can be broken down into
one class with RNA editing functions and a second class with ribonuclease functions. Related enzymes
function exclusively as amino hydrolases and are critical for various cellular functions including RNA editing,
antibody diversity, and viral defense. Each DYW-deaminase domains binds two zinc ions. Zinc #1 is most likely
coordinated by the typical C/HAE---CXXC motif found in all nucleotide deaminases. Amino acids in a unique C-
terminus likely coordinate Zinc #2 and are nearby the catalytic zinc ion binding site. A general mechanism for
cytidine deamination does not require two zinc ions. The second zinc ion could have a unique co-catalytic
function, serve as a structural feature, or have a role in ligand binding. Zinc ions are not required for all
ribonuclease mechanisms and addition of EDTA promotes the ribonuclease cleavage activity of recombinant
DYW-deaminases in vitro. One or both zinc ions might operate as a zinc “switch” that governs the deaminase
and ribonuclease functions of the domain.
This project aims to investigate a model where zinc ions act as a “switch” that determines catalytic
function. Zinc binding in DYW-deaminases with endoribonuclease and RNA editing functions will be compared
to investigate if amino acid differences present in each functional classes determine zinc stoichiometry. A pool
of recombinant DYW-deaminases with amino acid substitutions in putative zinc coordinating residues will be
created to pinpoint the location of zinc coordinating residues. Endoribonuclease activities will be assayed for
rDYW-deaminase proteins with known RNA editing functions as well as known endoribonuclease functions in
the presence or absence of metal chelators. Ribonuclease activity will be measured for recombinant proteins
with altered zinc stoichiometries and mutations in zinc coordinating residues. A series of proteins with amino
acid changes might discover a link between zinc binding and enzymatic function as a well as to establish the
reaction mechanism for RNA cleavage. An activity assay for RNA editing that includes addition of exogenous
DYW-deaminases will be developed. The activity of DYW-deaminases with different zinc stiochiometries will be
measured to determine the relationship between zinc ion binding and aminohydrolase activity. The second zinc
ion may have a specialized role in ligand binding and to test this possibility the nucleic acid ligand requirements
of the DYW-deaminase will be determined using RNAs and putative substrates with modified nucleotides.
Finally, crystallization conditions for recombinant proteins will be screened. Crystals could be used in future
experiments aimed at solving the structure of the DYW-deaminase.
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Michael Lloyd Hayes其他文献
Michael Lloyd Hayes的其他文献
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Engineering of PPR base editors to repair pathogenic SNPs at the level of RNA
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- 批准号:
10359636 - 财政年份:2021
- 资助金额:
$ 14.6万 - 项目类别:
The Role of Zinc ions for RNA Binding and Catalytic Function of the DYW-deaminase
锌离子对 RNA 结合的作用和 DYW 脱氨酶的催化功能
- 批准号:
9280248 - 财政年份:2017
- 资助金额:
$ 14.6万 - 项目类别:
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