Identification and characterization of mammalian mitochondrial tRNA nucleotidyltransferases.
The CCA-adding enzyme (ATP:tRNA adenylyltransferase or CTP:tRNA cytidylyltransferase (EC )) generates the conserved CCA sequence responsible for the attachment of amino acid at the 3' terminus of tRNA molecules. It was shown that enzymes from various organisms strictly recognize the elbow region of tRNA formed by the conserved D- and T-loops. However, most of the mammalian mitochondrial (mt) tRNAs lack consensus sequences in both D- and T-loops. To characterize the mammalian mt CCA-adding enzymes, we have partially purified the enzyme from bovine liver mitochondria and determined cDNA sequences from human and mouse dbESTs by mass spectrometric analysis. The identified sequences contained typical amino-terminal peptides for mitochondrial protein import and had characteristics of the class II nucleotidyltransferase superfamily that includes eukaryotic and eubacterial CCA-adding enzymes. The human recombinant enzyme was overexpressed in Escherichia coli, and its CCA-adding activity was characterized using several mt tRNAs as substrates. The results clearly show that the human mt CCA-adding enzyme can efficiently repair mt tRNAs that are poor substrates for the E. coli enzyme although both enzymes work equally well on cytoplasmic tRNAs. This suggests that the mammalian mt enzymes have evolved so as to recognize mt tRNAs with unusual structures.
Nagaike
T
T
Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Bldg. FSB-301, 5-1-5 Kashiwanoha, Kashiwa, Chiba Prefecture, 277-8562, Japan.
Suzuki
T
T
Tomari
Y
Y
Takemoto-Hori
C
C
Negayama
F
F
Watanabe
K
K
Ueda
T
T
D000595:Amino Acid Sequence; D000818:Animals; D002117:Calcitriol / Q000031:analogs & derivatives*; D002417:Cattle; D002893:Chromosomes, Human, Pair 3; D006801:Humans; D051379:Mice; D008930:Mitochondria, Liver / Q000201:enzymology*; D024101:Mitochondrial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D012316:RNA Nucleotidyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013379:Substrate Specificity
D024101:Mitochondrial Proteins; C113641:QW 1624F2-1; D012343:RNA, Transfer; D012316:RNA Nucleotidyltransferases; C023198:tRNA nucleotidyltransferase; D002117:Calcitriol