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Originally published In Press as doi:10.1074/jbc.M106202200 on August 14, 2001

J. Biol. Chem., Vol. 276, Issue 43, 40041-40049, October 26, 2001
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Identification and Characterization of Mammalian Mitochondrial tRNA nucleotidyltransferases*

Takashi NagaikeDagger , Tsutomu SuzukiDagger §, Yukihide Tomari§, Chie Takemoto-Hori||, Fumiko Negayama§, Kimitsuna WatanabeDagger §, and Takuya UedaDagger §

From the Dagger  Department of Integrated Biosciences, Graduate School of Frontier Sciences and § Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Bldg. FSB-301, 5-1-5 Kashiwanoha, Kashiwa, Chiba Prefecture, 277-8562, Japan and || RIKEN Genomic Science Center, Suehiro-cho 1-7-22, Tsurumi-ku, Yokohama-shi, Kanagawa 230-0045, Japan

The CCA-adding enzyme (ATP:tRNA adenylyltransferase or CTP:tRNA cytidylyltransferase (EC 2.7.7.25)) 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.


* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

All cDNA sequences encoding mammalian mitochondrial CCA-adding enzymes reported in this paper have been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession numbers AB063105, AB063106, and AB063107.

To whom correspondence should be addressed. Tel.: 81-471-36-5401; Fax: 81-471-36-3602; E-mail: t-suzuki@k.u-tokyo.ac.jp.


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.


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