Mutational Analysis of Fungal tRNA Nucleotidyltransferase
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Transfer RNA nucleotidyltransferase is an enzyme that is necessary for attaching a specific CCA nucleotide sequence to the 3' end of tRNA. What makes this enzyme unique is that it accomplishes this task without the aid of a template. Similar enzymes are found in many organisms including humans. The specific mechanism of this enzyme, however, is not clear. The project was concerned with performing specific mutations on the amino acid sequences of the fungal enzyme (from C. albicans). These mutations were targeted to residues thought to be involved with active site catalysis. The mutated enzymes were then expressed in E. Coli. The effects that these mutations had on enzyme activity were determined through activity assays. The majority of the mutations performed ablated enzymatic activity, demonstrating their importance to the CCA tRNA nucleotidyltransferase enzyme. One mutant retained activity, and was characterized. These mutations furthered understanding of the residues important for active site activity in C. Albicans CCA nucleotidyltransferase.