Characterization of a gene encoding tRNA nucleotidyltransferase from Candida glabrata

A gene encoding ATP (CTP):tRNA nucleotidyltransferase (EC2.7.7.25) was isolated from Candida (Torulopsis) glabrata by complementation in Saccharomyces cerevisiae. The predicted amino acid sequence of the protein revealed a large region with high sequence similarity to members of the Class II group of the nucleotidyltransferase superfamily and an N-terminal region characteristic of a mitochondrial targeting sequence. The essential role of the carboxylates within the conserved DXD and RRD motifs was confirmed by mutagenesis. C. glabrata strains bearing truncated CCA1 genes that lacked sequences encoding the putative mitochondrial targeting peptide were unable to grow on non-fermentable carbon sources but were able to grow on a fermentable carbon source. These results suggest that, as in S. cerevisiae, the C. glabrata CCA-adding enzyme is a sorting isozyme that functions in multiple cellular compartments. Mapping of the 5'-ends of primary transcripts of CCA1 revealed multiple transcription start sites located both upstream of and between two in-frame start codons. When the cells were cultured on a non-fermentable carbon source the longer transcripts appeared more abundant, suggesting that the choice of transcription start sites was influenced by carbon source. The shorter transcripts, which lacked sequences encoding the mitochondrial targeting information, were more predominant in cells grown on glucose. These observations suggest that expression of CCA-adding isozymes in C. glabrata may be regulated. The DNA sequence has been assigned GenBank Accession No. AF098803.