Mitochondrial (mt) tRNAMet has the unusual modified nucleotide 5-formylcytidine (f5C) in the first position of the anticodon. This tRNA must translate both AUG and AUA as methionine. By constructing an in vitro translation system from bovine liver mitochondria, we examined the decoding properties of the native mt tRNAMet carrying f5C in the anticodon compared to a transcript that lacks the modification. The native mt Met-tRNA could recognize both AUA and AUG codons as Met, but the corresponding synthetic tRNAMet lacking f5C (anticodon CAU), recognized only the AUG codon in both the codon-dependent ribosomal binding and in vitro translation assays. Furthermore, the Escherichia coli elongator tRNAMetm with the anticodon ac4CAU (ac4C = 4-acetylcytidine) and the bovine cytoplasmic initiator tRNAMet (anticodon CAU) translated only the AUG codon for Met on mt ribosome. The codon recognition patterns of these tRNAs were the same on E. coli ribosomes. These results demonstrate that the f5C modification in mt tRNAMet plays a crucial role in decoding the nonuniversal AUA codon as Met, and that the genetic code variation is compensated by a change in the tRNA anticodon, not by a change in the ribosome. Base pairing models of f5C-G and f5C-A based on the chemical properties of f5C are presented.

Animal mitochondrial protein synthesis systems contain two serine tRNAs (tRNAs Ser ) corresponding to the codons AGY and UCN, each possessing an unusual secondary structure; the former lacks the entire D arm, and the latter has a slightly different cloverleaf structure. To elucidate whether these two tRNAs Ser can be recognized by the single animal mitochondrial seryl-tRNA synthetase (mt SerRS), we purified mt SerRS from bovine liver 2400-fold and showed that it can aminoacylate both of them. Specific interaction between mt SerRS and either of the tRNAs Ser was also observed in a gel retardation assay. cDNA cloning of bovine mt SerRS revealed that the deduced amino acid sequence of the enzyme contains 518 amino acid residues. The cDNAs of human and mouse mt SerRS were obtained by reverse transcription-polymerase chain reaction and expressed sequence tag data base searches. Elaborate inspection of primary sequences of mammalian mt SerRSs revealed diversity in the N-terminal domain responsible for tRNA recognition, indicating that the recognition mechanism of mammalian mt SerRS differs considerably from that of its prokaryotic counterpart. In addition, the human mt SerRS gene was found to be located on chromosome 19q13.1, to which the autosomal deafness locus DFNA4 is mapped.

No abstract

No abstract