tRNA editing by deamination: Balancing affinity and specificity

核酸经历天然存在的化学修饰。超过100种不同 已经描述了修饰，并且嘌呤和嘧啶中的每个位置 碱基可以被修饰;糖通常也被修饰。尽管最近取得了进展， 大多数修饰的生物合成机制尚未完全了解，这是因为， 部分归因于与体外重建酶活性相关的困难。而 一些修饰可以用纯化的组分有效地形成，其它修饰可以 需要更复杂的路径一个修改相互依赖的模型，其中 一个修改是另一个的先决条件，可能解释了一个主要的障碍 在体外重建酶活性。这个模型是由我们早期的 在真核生物中发现tRNA胞嘧啶到尿苷编辑;一种反应， 在体外被概括，其机制仍然未知。形成 m3 C在体外的表达需要两者的存在，T.布鲁氏菌甲基转移酶TRM 140 和脱氨酶ADAT 2/3。一旦形成，m3 C通过相同的脱氨剂脱氨成m3 U。 一组酶。 这项拟议的研究在很大程度上依赖于我们确定具体的 TRM 140和ADAT 2/3的单个残基和结构域对 底物识别和催化。在我的实验室里，结合是由 电泳迁移率变化试验（EMSA），同时测定催化活性 依赖于基于孵育的甲基化测定的脱氨基， 放射性标记的底物与所讨论的酶。两者的结果 使用Typhoon型的 PhosphorImager系统。目前的补充是取代现有的单位， 它坏了，不能再用了。没有它， 这项研究几乎是不可能的 这一请求是与库尔特·弗雷德里克博士的一项提案同时提交的。 我在俄亥俄州州立大学的系也需要这种设备， 名为“精确核糖体易位的分子分析”的项目（R 01 GM 072528）。

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).