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).

核酸会经历 100 多种不同的化学修饰。修饰已被描述，并且嘌呤和嘧啶中的每个位置尽管最近取得了进展，但碱基可以被修饰；通常糖也被修饰。大多数修饰的生物合成机制尚未完全了解，因为，部分原因是与体外重建酶活性相关的困难。一些修饰可以用纯化的组分有效地形成，其他修饰可以需要更复杂的途径来修改相互依赖，其中一项修改是另一项修改的先决条件，可能解释一个主要障碍该模型是由我们早期提出的。发现真核生物中的 tRNA 胞嘧啶至尿苷编辑反应；其形成已在体外进行了重现，但其机制仍不清楚。体外 m3C 的产生需要 T. brucei 甲基转移酶 TRM140 的存在并且一旦形成m3C，脱氨酶ADAT2/3就被相同的脱氨酶脱氨基为m3U。一组酶。该研究建议在很大程度上依赖于我们确定具体目标的能力 TRM140 和 ADAT2/3 的各个残基和结构域的贡献在我的实验室中，底物识别和催化作用由下式确定。电泳迁移率变化测定 (EMSA)，同时测定催化活性依赖于基于孵化的甲基化检测的脱氨基作用两种酶的放射性标记底物的结果。使用台风型强制可视化和定量化验当前的补充是替换现有的 PhosphorImager 系统。已经损坏，无法再使用，没有它，就无法成功完成。所提出的研究几乎是不可能的该请求与 Kurt Fredrick 博士的提案同时提交。我在俄亥俄州立大学的系也需要这个设备题为“精确核糖体易位的分子分析”的项目（R01 GM072528）。

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）

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

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

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.

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