Abstract

The enzyme ATP(CTP): tRNA-specific tRNA nucleotidyltransferase catalyzes the addition of cytidine, cytidine, adenosine (CCA) to the 3’ end of transfer RNAs (tRNAs) during their synthesis and repair. This CCA sequence is required for aminoacylation and protein synthesis. The Saccharomyces cerevisiae enzyme is defined as a Class II tRNA nucleotidyltransferase through five motifs (A to E) conserved in the amino-terminal half of the protein. Functions have been assigned to each of these motifs. Our previous studies have shown that enzyme activity is reduced by mutations in motif C leading to a temperature-sensitive (ts) phenotype. Further, we also have shown that these ts mutations can be suppressed by a second-site mutation in motif A suggesting an interaction between motifs A and C. Here we use site-directed mutagenesis to make specific changes in residues in these motifs to explore these interactions.
Specifically, arginine 64 found in motif A was changed to phenylalanine, proline or alanine and the effects of these substitutions on wild-type and mutant backgrounds was determined. Changing arginine 64 to proline (R64P) resulted in a lethal phenotype in any background tested. Converting arginine to phenylalanine (R64F) resulted in no measurable changes in growth in a wild-type background in our in vivo assay and also suppressed the ts phenotype resulting from the conversion of glutamate 189 to phenylalanine (E189F) or lysine (as had been seen previously for an arginine 64 to tryptophan substitution). In contrast, the R64F substitution could not rescue the lethal phenotype resulting from conversion of aspartate 190 to phenylalanine (D190F). Changing arginine 64 to alanine (R64A) resulted in reduced growth at the restrictive temperature in a wild-type background and did not suppress the E189F, E189K, or D190A ts phenotypes. Moreover, the R64A change in the D190A background enhanced the growth defect at the permissive temperature. Taken together, these data support our hypothesis of a direct interaction between elements of motifs A and C. Computational modelling is used to explore how the phenotypes observed are linked to changes in the structure of the protein.