Novel RNA catalysts fortRNA aminoacylation
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tRNA aminoacylation is an important step during protein synthesis. This reaction is catalyzed by a group of enzymes called the aminoacyl tRNA synthetases (ARSs). There are 20 ARSs, one for each of the 20 amino acids in most organisms. The ARSs are sophisticated proteins that could not have existed before protein synthesis machinery was established. This leads to the question: how was tRNA aminoacylation catalyzed prior to the emergence of the ARSs? One possible answer is that tRNA aminoacylation was catalyzed by RNA. This notion is based on the RNA world hypothesis, which proposes that during the development of early life, and before the emergence of the protein-based life, RNA was involved in information storage and catalysis, and in the emergence of a protein-based life. The search for RNA catalysts (ribozymes) for tRNA aminoacylation has yielded an ambidextrous ribozyme, AD02 that can transfer glutamine to the 3 ' -end of a tRNA. This study describes my attempts to expand the repertoire of nbozymes that recognize other amino acids, in particular, leucine. The most active ribozyme obtained in this study could transfer leucine to its own 5 ' -hydroxyl group. This ribozyne is a trans -aminoacylation catalyst. It forms a pseudoknot structure in its active site in a manner similar to that of AD02. A comparison of the properties of AD02 and KL17 reveals that such RNA catalysts might have existed in a primitive translation machinery. In addition to providing evidence for the RNA world hypothesis, ribozymes that aminoacylate tRNA have technological applications. In particular they can be used to generate nonnatural aminoacyl-tRNAs. One such ribozyme is Flexizyme, which can aminoacylate various tRNAs with nonnatural derivatives of phenylalanine. However, this ribozyme would be more useful if it exhibits tRNA specificity. To achieve this we appended a tRNA specificity sequence to its 3 ' -end and generated a family of designer ribozymes. Each ribozyme in this family specifically aminoacylates its cognate tRNA, using positive and negative determinants. Another study was undertaken to obtain ribozymes that catalyze peptidyl transferase reaction. However this study yielded aptamers (an RNA molecule that binds to a specific ligand) to the tRNA substrates.