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We report that the temperature-sensitive (ts) phenotype in Saccharomyces cerevisiae associated with a variant tRNA nucleotidyltransferase containing an amino acid substitution at position 189 results from a reduced ability to incorporate AMP and CMP into tRNAs. We show that this defect can be compensated for by a second-site suppressor converting residue arginine 64 to tryptophan. The R64W substitution does not alter the structure or thermal stability of the enzyme dramatically but restores catalytic activity in vitro and suppresses the ts phenotype in vivo. R64 is found in motif A known to be involved in catalysis and nucleotide triphosphate binding while E189 lies within motif C previously thought only to connect the head and neck domains of the protein. Although mutagenesis experiments indicate that residues R64 and E189 do not interact directly, our data suggest a critical role for residue E189 in enzyme structure and function. Both R64 and E189 may contribute to the organization of the catalytic domain of the enzyme. These results, along with overexpression and deletion analyses, show that the ts phenotype of cca1-E189F does not arise from thermal instability of the variant tRNA nucleotidyltransferase but instead from the inability of a partially active enzyme to support growth only at higher temperatures.

Original publication

DOI

10.1016/j.bbapap.2013.07.003

Type

Journal article

Journal

Biochim Biophys Acta

Publication Date

10/2013

Volume

1834

Pages

2097 - 2106

Keywords

Hypomorphic, Motif C, Suppressor, Temperature-sensitive, Yeast, tRNA nucleotidyltransferase, Adenosine Monophosphate, Amino Acid Motifs, Amino Acid Substitution, Arginine, Aspartic Acid, Catalytic Domain, Cytidine Monophosphate, Hot Temperature, Molecular Dynamics Simulation, Molecular Sequence Data, Phenotype, Protein Structure, Secondary, RNA Nucleotidyltransferases, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Sequence Alignment, Tryptophan