Identification and Characterization of Mammalian Mitochondrial tRNA nucleotidyltransferases

Takashi Nagaike,Tsutomu Suzuki,Yukihide Tomari,Chie Takemoto-Hori,Fumiko Negayama,Kimitsuna Watanabe,Takuya Ueda

doi:10.1074/jbc.m106202200

Abstract

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.

Highlights

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
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 both enzymes work well on cytoplasmic tRNAs
The results clearly showed that the human mt CCA-adding enzyme efficiently repaired the mt tRNAs whereas the E. coli enzyme had a much lower efficiency, both enzymes work well on cytoplasmic tRNAs

Summary

EXPERIMENTAL PROCEDURES

Materials [␣-32P]ATP (3000 Ci/mmol) and [␣-32P]CTP (3000 Ci/mmol) were obtained from Amersham Pharmacia Biotech. Step 3—The pooled fractions were applied to a SP-Sepharose fast flow column (1.6 ϫ 10 cm) equilibrated with HG. and developed with a linear gradient (20 –300 mM KCl in Buffer HG) at a flow rate of 1.5 ml/min. Step 5—The sample was applied to a hydroxyapatite column (BioScale CHT2-I (2 ml)) equilibrated with PG. and developed with a linear gradient from 100 to 300 mM potassium phosphate in Buffer PG at a flow rate of 0.5 ml/min. Following SDS-PAGE, candidate bands were excised, soaked in a buffer containing 0.2 M NH4HCO3 with 50% acetonitrile, and incubated at 30 °C for 30 min to remove SDS from the gels. The collected fractions were combined, and the peptides were dried and dissolved in 40 ␮l of 0.1% formic acid for peptide mass mapping by LC/MS/MS

Mass Spectrometry and Protein Identification

Expression and Purification of the Recombinant Human Enzyme

Determination of Km and kcat Values

RESULTS

DISCUSSION

Substrate mt tRNAAla yeast tRNAPhe