In vivo analysis of mutated initiation codons in the mitochondrial COX2 gene of Saccharomyces cerevisiae fused to the reporter gene ARG8(m) reveals lack of downstream reinitiation

To examine normal and aberrant translation initiation in Saccharomyces cere visiae mitochondria, we fused the synthetic mitochondrial reporter gene ARG 8(m) to codon 91 of the COX-2 coding sequence and inserted the chimeric gen e into mitochondrial DNA (mtDNA). Translation of the cox2(1-91)::ARG8(m) mR NA yielded a fusion protein precursor that was processed to yield wild-type Arg8p. Thus mitochondrial translation could be monitored by the ability of mutant chimeric genes to complement a nuclear arg8 mutation. As expected, translation of the cox2(1-91)::ARG8(m) mRNA was dependent on the COX2 mRNA- specific activator PET111. We tested the ability of six triplets to functio n as initiation codons in both the cox2(1-91)::ARG8(m) reporter mRNA and th e otherwise wild-type COX2 mRNA. Substitution of AUG, CCC or AAA for the in itiation codon abolished detectable translation of both mRNAs, even when PE T111 activity was increased. The failure of these mutant cox2(1-91)::ARG8(m ) genes to yield Arg8p demonstrates that initiation at downstream AUG codon s, such as COX2 codon 14, does not occur even when normal initiation is blo cked. Three mutant triplets at the site of the initiation codon supported d etectable translation, with efficiencies decreasing in the order GUG, AUU, AUA. increased PET111 activity enhanced initiation at AUU and AUA codons. C omparisons of expression, at the level of accumulated product, of cox2(1-91 )::ARG8(m) and COX2 carrying these mutant initiation codons revealed that v ery low-efficiency translation can provide enough Cox2p to sustain signific ant respiratory growth, presumably because Cox2p is efficiently assembled i nto stable cytochrome oxidase complexes.