Abstract

Mature tRNAs require a 3'-terminal cytidine-cytidine-adenosine sequence to function in protein synthesis. The addition of these nucleotides into tRNAs with incomplete 3'-termini is catalyzed by the enzyme tRNA nucleotidyltransferase. Genetic studies have resulted in the isolation of the gene coding for tRNA nucleotidyltransferase in yeast and in vivo studies identified a temperature-sensitive yeast strain with a mutant CCA1 gene, which can survive at room temperature, but not at 37{493}C. Gene sequencing revealed that the temperature-sensitive phenotype came from a single nucleotide substitution in the CCA1 gene, which led to the substitution of a glutamate residue by a lysine in the protein (D. Kushner, personal communication). In this work, enzymatic characterization showed that this lysine variant had approximately 5% of the activity of the wild-type enzyme and a tertiary structural change as detected by fluorescence spectroscopy at the permissive temperature. Thermal stability monitored by circular dichroism showed that the lysine variant is less thermally stable than the wild-type enzyme with an approximately 6{493}C lower transitional temperature.