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tRNA editing by deamination: Balancing affinity and specificity

通过脱氨基进行 tRNA 编辑：平衡亲和力和特异性

基本信息

批准号：
10389330
负责人：
Juan D Alfonzo
金额：
$ 7.23万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-08-01 至 2023-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10389330
关键词：
12 year old Affect Affinity Anabolism Anticodon Arginine Specific tRNA Binding Biochemical Biological Assay Catalysis Cell Nucleus Cells Chemicals Codon Nucleotides Cytosine Deaminase Deamination Deuterium Electrophoretic Mobility Shift Assay Enzymes Equilibrium Equipment Eukaryota Funding Gene Expression Genetic Transcription Genome Genomics Human In Vitro Individual Label Laboratories Leishmania Luciferases Maps Mass Spectrum Analysis Mediating Medical Methylation Methyltransferase Modeling Modification Molecular Analysis Mutagenesis National Institute of General Medical Sciences Nuclear Nucleic Acids Ohio Organism Pathway interactions Positioning Attribute Proteins Proteomics Purines Pyrimidine RNA Recognition Motif Radioactive Reaction Regulation Reporter Research Ribosomes Services Site Specific qualifier value Specificity System Techniques Testing Threonine-Specific tRNA Transfer RNA Translations Trypanosoma Trypanosoma brucei brucei Universities Untranslated Regions Uridine Variant Visualization base enzyme activity in vitro activity in vivo instrument parent grant reconstitution ribosome profiling sugar transcriptome

项目摘要

Nucleic acids undergo naturally occurring chemical modifications. Over 100 different modifications have been described and every position in the purine and pyrimidine bases can be modified; often the sugar is also modified. Despite recent progress, the mechanism for the biosynthesis of most modifications is not fully understood, owing, in part, to the difficulty associated with reconstituting enzyme activity in vitro. Whereas some modifications can be efficiently formed with purified components, others may require more intricate pathways. A model for modification interdependence, in which one modification is a prerequisite for another, potentially explains a major hindrance in reconstituting enzymatic activity in vitro. This model was prompted by our earlier discovery of tRNA cytosine-to-uridine editing in eukaryotes; a reaction that had not been recapitulated in vitro and the mechanism for which remains unknown. Formation of m3C in vitro requires the presence of both, the T. brucei methyltransferase TRM140 and the deaminase ADAT2/3. Once formed m3C is deaminated to m3U by the same set of enzymes. The propose research relies heavily on our ability to determine the specific contributions of individual residues and domains of TRM140 and ADAT2/3 to substrate recognition and catalysis. Binding in my laboratory is determined by Eletrophoretic Mobility Shift Assays (EMSA), while determination of catalytic activity relies on either deamination of methylation assays based on incubation a radioactively labeled substrate with the enzyme(s) in question. The results of both assays are forcibly visualized and quantified by using a Typhoon-type PhosphorImager system. The current supplement is to replace an existing unit which has broken down and it is no longer serviceable. Without it, successful completion of the research proposed is nearly impossible This request is being submitted in parallel with a proposal from Dr. Kurt Fredrick in my department at Ohio State University who also requires this equipment for his project entitled “Molecular analysis of accurate ribosomal translocation” (R01 GM072528).

核酸经过自然发生的化学修饰。超过100种不同的

项目成果

期刊论文数量（42）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

From Prebiotics to Probiotics: The Evolution and Functions of tRNA Modifications.

DOI：
10.3390/life6010013
发表时间：
2016-03-14
期刊：
Life (Basel, Switzerland)
影响因子：
0
作者：
McKenney KM;Alfonzo JD
通讯作者：
Alfonzo JD

RNAi, the guiding principle and keeping family happy.

RNAi，指导原则和保持家庭幸福。

DOI：
10.1261/rna.049635.115
发表时间：
2015
期刊：
RNA (New York, N.Y.)
影响因子：
0
作者：
Alfonzo,JuanD
通讯作者：
Alfonzo,JuanD

How the intracellular partitioning of tRNA and tRNA modification enzymes affects mitochondrial function.

DOI：
10.1002/iub.1957
发表时间：
2018-12
期刊：
IUBMB life
影响因子：
4.6
作者：
Paris Z;Alfonzo JD
通讯作者：
Alfonzo JD

Binding synergy as an essential step for tRNA editing and modification enzyme codependence in Trypanosoma brucei.

DOI：
10.1261/rna.062893.117
发表时间：
2018-01
期刊：
RNA (New York, N.Y.)
影响因子：
0
作者：
McKenney KM;Rubio MAT;Alfonzo JD
通讯作者：
Alfonzo JD

Unusual noncanonical intron editing is important for tRNA splicing in Trypanosoma brucei.

DOI：
10.1016/j.molcel.2013.08.042
发表时间：
2013-10-24
期刊：
MOLECULAR CELL
影响因子：
16
作者：
Rubio, Mary Anne T.;Paris, Zdenek;Gaston, Kirk W.;Fleming, Ian M. C.;Sample, Paul;Trotta, Christopher R.;Alfonzo, Juan D.
通讯作者：
Alfonzo, Juan D.