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To delineate the total number of tRNAs encoded by yeast mitochondrial DNA (mtDNA), we have examined mitochondrial tRNA preparations for the presence of heterogenic isoaccepting tRNAs. Analyses of /sup 3/H-labeled aminoacylated mitochondrial tRNAs by reversed-phase column chromatography (RPC-5) coupled with hybridization to mtDNA detected only one major mitochondrially coded tRNA for alanine, arginine, aspartic acid, glycine, histidine, isoleucine, leucine, lysine, proline, serine, and tryptophan. Some of these profiles also contained one or more minor peaks that may represent small amounts of heterogenic isoacceptors, but their low concentrations prevented their characterization. Cysteinyl-, methionyl-, phenylalanyl-, threonyl-, tyrosyl-, and valyl-tRNAs separated into multiple species upon RPC-5 chromatography. Two cysteinyl-, two methionyl-, two phenylalanyl-, two threonyl-, four tyrosyl-, and two valyl-tRNA species hybridized to mtDNA. The hybridization of the phenylalanyl- and the valyl-tRNA species was not additive, indicating that their sequences are similar, if not identical, and suggesting that they may be transcribed from the same genes. The two methionyl-, and two threonyl-, and probably the two cysteinyl-tRNAs are transcribed from separate genes, since their hybridizations to mtDNA are additive. That the methionyl-tRNAs are transcribed from separate genes was further confirmed through deletion mapping experiments which showed that the genes coding for these tRNAsmore » are at different locations on the mtDNA. The transcriptional relationship of the four tyrosyl-tRNAs was not established. There is at least a cistron coding for tRNAs corresponding to each of the common amion acids except asparagine and several amino acids (methionine, threonine, and cysteine) are accepted by more than one transcriptionally distinct tRNA. Thus, a minimum of 22 tRNA cistrons have been identified.« less

We have previously identified 14 aminoacyl tRNAs that are specified by yeast mitochondrial DNA (mtDNA). We now report four more amino acids (Arg, Cys, Trp, Thr) that acylate tRNAs which hybridize with mtDNA. Furthermore one of the two mitochondrial tRNAs that we had earlier demonstrated to be directly charged with glutamic acid responds to glutamine but not to glutamic acid codons. Thus Gln-tRNAGln appears to be formed by transamidation of a missense intermediate Glu-tRNAGln. This brings to 19 the number of amino acids which have corresponding tRNAs specified by mtDNA. Only tRNAAsn has not yet been shown to be a mtDNA transcript. We have also mapped the genes for the newly identified mitochondrial tRNAs, as well as several others that were previously identified but unmapped, by hybridization to the mtDNA of a series of petite deletion mutants. We now have ordered 20 mitochondrial tRNA genes (including two methionyl-tRNAs) wtih respect to the antibiotic resistance markers chloramphenicol (CR), erythromycin (ER), paromomycin (PR), and oligomycin I and II (ORI, ORII). Eighteen tRNA genes map between the C and E resistance markers. Only the serinyl-tRNA and glutamyl-tRNA genes are localized near the OI and OII resistance markers.