tRNA editing by deamination: Balancing affinity and specificity

核酸经历自然发生的化学修饰。100多种不同的 已经描述了修饰，以及嘌呤和嘧啶中的每个位置 碱基可以被修饰；通常糖也可以被修饰。尽管最近取得了进展，但 大多数修饰的生物合成机制尚未完全了解，由于， 这在一定程度上是由于在体外重建酶活性的困难。鉴于 一些修饰可以用纯化的组分有效地形成，其他的可以 需要更复杂的路径。一种修改相互依赖模型，其中 一项修改是另一项修改的先决条件，这可能解释了一个主要障碍 在体外重建酶的活性。这个模型是由我们早先提出的 在真核生物中发现tRNA胞嘧啶到尿苷的编辑；一种尚未发生的反应 已在体外进行了概括，其机制尚不清楚。形成 在体外M3C需要两者的存在，布氏毛滴虫甲基转移酶TRM140 和脱氨酶ADAT2/3。一旦形成，M3C通过相同的脱氨酶被脱氨为M3U 一套酶。 提议的研究在很大程度上依赖于我们确定具体 TRM140和ADAT2/3的单个残基和结构域对 底物识别和催化。我实验室里的绑定是由以下因素决定的 电迁移分析(EMSA)，同时测定催化活性 依赖于基于孵化的甲基化分析的脱氨基 用有问题的酶(S)放射性标记底物。两者的结果都是 通过使用台风类型强制可视化和量化化验 荧光成像仪系统。目前的补充方案是取代现有的一个单位，该单位 已经坏了，不能再使用了。如果没有它，成功地完成 提出的研究几乎是不可能的。 这一请求与库尔特·弗雷德里克博士于#年提出的一项建议同时提交 我在俄亥俄州立大学的系也需要这种设备 题为“准确核糖体易位的分子分析”的项目(R01 GM072528)。

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).