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 CCA-adding enzyme adds and repairs the conserved CCA sequence of the 3Ј terminus of tRNA using CTP and ATP as substrates. The CCA terminus of the tRNA molecule is the attachment site for the amino acid, and most of the aminoacyltRNA synthetases and elongation factor Tu require this sequence to be present to function (1-3). Furthermore, it has been shown that the CCA sequence is necessary for the exact positioning of the peptidyl-tRNA at the P site and aminoacyl-tRNA at the A site on the large ribosomal subunit to facilitate peptide bond formation (4 -6). In certain organisms such as eukaryotes, some archaea, and many eubacteria, the tRNA genes do not encode the CCA sequence; therefore its addition is an essential step for tRNA maturation (7).CCA-adding enzymes belong to the nucleotidyltransferase superfamily, which is divided into two classes (8). Class I contains the archaeal CCA-adding enzyme and eukaryotic poly(A) polymerases, whereas class II contains eubacterial and eukaryotic CCA-adding enzymes and eubacterial poly(A) polymerases. The class II CCA-adding enzymes exhibit significant homology in the over 25-kDa region including the active site, which commonly has DXD and RRD motifs (8), whereas class I enzymes do not show a significant homology with class II enzymes around the active site.The addition of CCA by the CCA-adding enzymes does not require any nucleic acid template unlike other DNA or RNA polymerases. Several mechanisms for CCA addition have been hypothesized from the results of biochemical experiments (9 -11). We proposed the "dead-end AMP incorporation hypothesis" based on the finding that the class II enzymes have significantly high affinity for ATP ...

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Background: Metazoan mitochondrial (mt) tRNAs are structurally quite different from the canonical cloverleaf secondary structure. The mammalian mt tRNA Ser GCU for AGY codons (Y C or U) lacks the entire D arm, whereas tRNA Ser UGA for UCN codons (N A, G, C or U) has an extended anti-codon stem. It has been a long-standing problem to prove experimentally how these tRNAs Ser work in the mt translation system.

The mammalian mitochondrial ribosome (mitoribosome) has a highly protein-rich composition with a small sedimentation coefficient of 55 S, consisting of 39 S large and 28 S small subunits. In the previous study, we analyzed 39 S large subunit proteins from bovine mitoribosome (Suzuki, T., Terasaki, M., Takemoto-Hori, C., Hanada, T., Ueda, T., Wada, A., and Watanabe, K. (2001) J. Biol. Chem. 276, 21724-21736). The results suggested structural compensation for the rRNA deficit through proteins of increased molecular mass in the mitoribosome. We report here the identification of 28 S small subunit proteins. Each protein was separated by radical-free high-reducing two-dimensional polyacrylamide gel electrophoresis and analyzed by liquid chromatography/mass spectrometry/mass spectrometry using electrospray ionization/ion trap mass spectrometer to identify cDNA sequence by expressed sequence tag data base searches in silico. Twenty one proteins from the small subunit were identified, including 11 new proteins along with their complete cDNA sequences from human and mouse. In addition to these proteins, three new proteins were also identified in the 55 S mitoribosome. We have clearly identified a mitochondrial homologue of S12, which is a key regulatory protein of translation fidelity and a candidate for the autosomal dominant deafness gene, DFNA4. The apoptosis-related protein DAP3 was found to be a component of the small subunit, indicating a new function for the mitoribosome in programmed cell death. In summary, we have mapped a total of 55 proteins from the 55 S mitoribosome on the two-dimensional polyacrylamide gels.