In vivo analysis of mutated initiation codons in the mitochondrial COX2 gene of Saccharomyces cerevisiae fused to the reporter gene ARG8m reveals lack of downstream reinitiation.
To examine normal and aberrant translation initiation in Saccharomyces cerevisiae mitochondria, we fused the synthetic mitochondrial reporter gene ARG8m to codon 91 of the COX2 coding sequence and inserted the chimeric gene into mitochondrial DNA (mtDNA). Translation of the cox2(1-91)::ARG8m mRNA 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)::ARG8m mRNA was dependent on the COX2 mRNA-specific activator PET111. We tested the ability of six triplets to function as initiation codons in both the cox2(1-91)::ARG8m reporter mRNA and the otherwise wild-type COX2 mRNA. Substitution of AUC, CCC or AAA for the initiation codon abolished detectable translation of both mRNAs, even when PET111 activity was increased. The failure of these mutant cox2(1-91)::ARG8m genes to yield Arg8p demonstrates that initiation at downstream AUG codons, such as COX2 codon 14, does not occur even when normal initiation is blocked. Three mutant triplets at the site of the initiation codon supported detectable translation, with efficiencies decreasing in the order GUG, AUU, AUA. Increased PET111 activity enhanced initiation at AUU and AUA codons. Comparisons of expression, at the level of accumulated product, of cox2(1-91)::ARG8m and COX2 carrying these mutant initiation codons revealed that very low-efficiency translation can provide enough Cox2p to sustain significant respiratory growth, presumably because Cox2p is efficiently assembled into stable cytochrome oxidase complexes.
Bonnefoy
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Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853-2703, USA.
Fox
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D000595:Amino Acid Sequence; D019677:Artificial Gene Fusion; D001483:Base Sequence; D018387:Codon, Initiator / Q000235:genetics*; D051546:Cyclooxygenase 2; D004271:DNA, Fungal / Q000235:genetics; D004272:DNA, Mitochondrial / Q000235:genetics; D005800:Genes, Fungal; D017930:Genes, Reporter; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics*; D009154:Mutation; D010442:Peptide Chain Initiation, Translational / Q000235:genetics; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis / Q000235:genetics*; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D000637:Transaminases / Q000096:biosynthesis / Q000235:genetics
D018387:Codon, Initiator; D004271:DNA, Fungal; D004272:DNA, Mitochondrial; D007527:Isoenzymes; D012331:RNA, Fungal; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D000637:Transaminases; C018731:acetylornithine transaminase