REDUCED BUT ACCURATE TRANSLATION FROM A MUTANT AUA INITIATION CODON IN THE MITOCHONDRIAL COX2 MESSENGER-RNA OF SACCHAROMYCES-CEREVISIAE

We have changed the translation initiation codon of the COX2 mRNA of S accharomyces cerevisiae from AUG to AUA, generating a mutation termed cox2-10. This mutation reduced translation of the COX2 mRNA at least f ive-fold without affecting the steady-state level of the mRNA, and pro duced a leaky nonrespiratory growth phenotype. To address the question of whether residual translation of the cox2-10 mRNA was initiating at the altered initiation codon or at the next AUG codon downstream (at position 14), we took advantage of the fact that the mature coxII prot ein is generated from the electrophoretically distinguishable coxII pr ecursor by removal of the amino-terminal 15 residues, and that this pr ocessing can be blocked by a mutation in the nuclear gene PET2858. We constructed a pet2858, cox2-10 double mutant strain using a pet2858 al lele from our mutant collection. The double mutant accumulated low lev els of a polypeptide which comigrated with the coxII precursor protein , not the mature species, providing strong evidence that residual init iation was occurring at the mutant AUA codon. Residual translation of the mutant mRNA required the COX2 mRNA-specific activator PET111. Furt hermore, growth of cox2-10 mutant strains was sensitive to alterations in PET111 gene dosage: the respiratory-defective growth phenotype was partially suppressed in haploid strains containing PET111 on a high-c opy-number vector, but became more severe in diploid strains containin g only one functional copy of PET111.