Fig. 5. Secondary structure of the rRNA decoding site in yeast mitochondria. (A) The ribosomal decoding site of Saccharomyces cerevisiae mitochondrial ribosomes; rRNA residues are numbered according to the position in yeast mtDNA. (B) Mitochondrial mutant decoding site carrying a cytosine-to-guanine mutation at position 1477 (corresponding to E. coli position 1409). Note that yeast mitochondrial rRNA position 1477 is not homologous to human mitochondrial rRNA position 1494 but to position 1493 (compare with Fig. 1). In addition, the lower stem of the yeast mitochondrial rRNA helix resembles its bacterial homologue helix 44 rather than its human mitochondrial counterpart: e.g., a Watson-Crick interaction at yeast mitochondrial rRNA positions C1477-G1583 (bacterial C1409-G1491) and G1478-C1582 (bacterial G1410-C1490), the corresponding human mt rRNA positions C1493•C1556 and C1494•A1555 are no Watson-Crick interactions.

Fig. 6. Capacity and accuracy of translation in mitochondrial hybrid and C1556G ribosomes. AUG(UUU)₁₂ mRNA-directed incorporation of [¹⁴C]-phenylalanine (○) and [³H]leucine (■) in a cell-free translation assay (mean ± SD; n = 3). (A) Bacterial hybrid ribosomes with a wild-type mitochondrial decoding site. (B) Mutant mitochondrial hybrid ribosomes with a cytosine-toguanine alteration at position 1491 (mtDNA 1556), which creates a canonical C-G base pair interaction. The ratio of leucine per phenylalanine incorporation is given in Table 1.

Fig. 7. To distinguish misincorporation from premature termination we analyzed ³⁵S-methionine-labeled proteins synthesized in cell-free translation assays by purified bacterial and mitochondrial hybrid ribosomes by using SDS/PAGE (12%) fluorography. Mutant A1555G and wild-type mitochondrial hybrid ribosomes produced the same level of full-length proteins, as assessed by translation of luciferase mRNA in a coupled transcription-translation reaction and by translation of L1 mRNA in T7-mRNA driven translation. (A) Proteins translated during luciferase coupled transcription-translation showing full-length luciferase (61 kDa), truncated luciferase transcribed from an internal start codon (48 KDa), and b-lactamase (31.5 kDa). (B) T7-mRNA-driven synthesis of Methanococcus jannaschii ribosomal protein L1 (25 kDa).

Fig. 8. Comparison of the mitochondrial A1555G mutant with the bacterial G1491C mutant. (A) Structural comparison of the base-pairing pattern in the ribosomal A site. Both the A1555G and the G1491C mutant are characterized by a C•C opposition at position 1409-1491 (mitochondrial 1493-1556, respectively) and an adjacent C-G Watson-Crick pair. (B) Translation phenotypes of wild-type and mutant bacterial and mitochondrial decoding sites. The amount of [¹⁴C]-phenylalanine and [³H]-leucine incorporated in a AUG(UUU)₁₂ mRNA-driven translation assay is presented.