In-vitro translation of mitochondrial mRNAs by yeast mitochondrial ribosomes is hampered by the lack of start-codon recognition

Summary In an attempt to reconstitute an homologous in-vitro translation system for yeast mitochondrial mRNAs, we have isolated ribosomes, supernatant factors, and tRNAs from mitochondria of Saccharomyces carlsbergensis. While poly(U) is translated faithfully in this system, no translation of in-vitro synthesised cytochrome c oxidase subunit II (COX2) mRNA could be detected. Formation of formylmethionyl-puromycin on mitochondrial ribosomes is stimulated by ApUpG, but not by COX2 mRNA, although mitochondrial small ribosomal subunits bind to this mRNA in vitro, even without added tRNA and initiation factors. We conclude, therefore, that the inability to faithfully translate mitochondrial mRNAs in vitro may be the result of an inability of mitochondrial ribosomes to recognize the initiation codon.