A point mutation in the 5′-untranslated leader that affects translational activation of the mitochondrial COX 3 mRNA

Abstract

The 613-base 5′-untranslated leader (5′-UTL) of the Saccharomyces cerevisiae mitochondrial COX 3 mRNA contains the target of an mRNA-specific translational activator complex composed of at least three nuclearly encoded proteins. We have genetically mapped a collection of cox 3 point mutations, using a set of defined COX 3 deletions, and found one to be located in the region coding the 5′-UTL. The strain carrying this allele was specifically defective in translation of the COX 3 mRNA. Nucleotide-sequence analysis showed that the allele was in fact a double mutation comprised of a single-base insertion in the 5′-UTL (T inserted between bases-428 and-427 with respect to the start of translation) and a G to A substitution at+3 that changed the ATG initiation codon to ATA. Both mutations were required to block translation completely. The effects of the ATG to ATA mutation alone (cox 3-1) had previously been analyzed in this laboratory: it reduces, but does not eliminate, translation, causing a slow respiratory growth phenotype. The T insertion in the 5′-UTL had no detectable respiratory growth phenotype as a single mutation. However, the 5′-UTL insertion mutation enhanced the respiratory defective phenotype of missense mutations in pet 54, one of the COX 3-specific translational-activator genes. This phenotypic enhancement suggests that the-400 region of the 5′-UTL, where the mutation is located, is important for Pet54p-COX 3 mRNA interaction.