A POINT MUTATION IN THE 5'-UNTRANSLATED LEADER THAT AFFECTS TRANSLATIONAL ACTIVATION OF THE MITOCHONDRIAL COX-3 MESSENGER-RNA

The 613-base 5'-untranslated leader (5'-UTL) of the Saccharomyces cere visiae mitochondrial COX3 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 cox3 poin t mutations, using a set of defined COX3 deletions, and found one to b e located in the region coding the 5'-UTL. The strain carrying this al lele was specifically defective in translation of the COX3 mRNA. Nucle otide-sequence analysis showed that the allele was in fact a double mu tation 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 (cox3-1) had previously b een analyzed in this laboratory: it reduces, but does not eliminate, t ranslation, causing a slow respiratory growth phenotype. The T inserti on in the 5'-UTL had no detectable respiratory growth phenotype as a s ingle mutation. However, the 5'-UTL insertion mutation enhanced the re spiratory defective phenotype of missense mutations in pet54, one of t he COXS-specific translational-activator genes. This phenotypic enhanc ement suggests that the -400 region of the 5'-UTL, where the mutation is located, is important for Pet54p-COX3 mRNA interaction.