ORIGINAL PAPER

Molecular and General Genetics MGG

, Volume 262, Issue 6, pp 1036-1046

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

  • N. BonnefoyAffiliated withDepartment of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853-2703, USA E-mail: tdf1@cornell.edu
  • , T. D. FoxAffiliated withDepartment of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853-2703, USA E-mail: tdf1@cornell.edu

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Abstract

To examine normal and aberrant translation initiation in Saccharomyces cerevisiae mitochondria, we fused the synthetic mitochondrial reporter gene ARG8 m to codon 91 of the COX2 coding sequence and inserted the chimeric gene into mitochondrial DNA (mtDNA). Translation of the cox2(1–91)::ARG8 m 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)::ARG8 m 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)::ARG8 m 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)::ARG8 m 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)::ARG8 m 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.

Key wordsSaccharomyces cerevisiae Mitochondrial translation Translation initiation Downstream initiation