Isolation and nucleotide sequence of a gene encoding tRNA nucleotidyltransferase from Kluyveromyces lactis.
A gene (KlCCA1) encoding ATP(CTP):tRNA specific tRNA nucleotidyltransferase (EC 2.7.7.25) was isolated from Kluyveromyces lactis by complementation of the Saccharomyces cerevisiae cca1-1 mutation. Sequencing of a 2665 bp EcoRI-SpeI restriction fragment revealed an open reading frame potentially encoding a protein of 489 amino acids with 57% sequence similarity to its S. cerevisiae homologue. Southern hybridization revealed a single copy of KlCCA1 in the K. lactis genome. KlCCA1 was able to complement both the mitochondrial and cytosolic defects in the cca1-1 mutant, suggesting that, as in S. cerevisiae, the K. lactis gene encodes a sorting isozyme that is targeted to mitochondria and the nucleus and/or cytosol. An altered KlCCA1 gene encoding a tRNA nucleotidyltransferase that lacked its first 35 amino acids was able to complement the nuclear/cytosolic but not the mitochondrial defect in the S. cerevisiae cca1-1 mutant, suggesting that the 35 amino-terminal amino acids are necessary for targeting to mitochondria but are not required for enzyme activity. Our results suggest that the mechanisms for production and distribution of mitochondrial and nuclear/cytosolic tRNA nucleotidyltransferase in K. lactis differ from those seen in S. cerevisiae.
Deng
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Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec, Canada.
Hanic-Joyce
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Joyce
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002467:Cell Nucleus / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D007716:Kluyveromyces / Q000201:enzymology* / Q000235:genetics*; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D009154:Mutation; D012316:RNA Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D017422:Sequence Analysis, DNA
D012316:RNA Nucleotidyltransferases; C023198:tRNA nucleotidyltransferase