tRNA editing by deamination: Balancing affinity and specificity

核酸经过自然发生的化学修饰。超过100种不同的

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).