ANALYSIS OF THE SACCHAROMYCES-CEREVISIAE MITOCHONDRIAL COX3 MESSENGER-RNA 5' UNTRANSLATED LEADER - TRANSLATIONAL ACTIVATION AND MESSENGER-RNA PROCESSING

We used transformation of yeast mitochondria and homologous gene repla cement to study features of the 613-base COX3 mRNA 5' untranslated lea der (5'-UTL) required for translational activation by the protein prod ucts of the nuclear genes PET54, PET122, and PET494 in vivo. Eliminati on of the single AUG triplet in the 5'-UTL had no detectable effect on expression, indicating that activator proteins do not work by allowin g ribosomes to bypass that AUG. Deletion of the entire 5'-UTL complete ly prevented translation, suggesting that the activator proteins do no t function by antagonizing any other negative element in the 5'-UTL. R emoval of the 15 terminal bases from the 5' end of the 5'-UTL did not block activator dependent translation. The largest internal deletion t hat did not interfere with translation removed 125 bases from the upst ream portion of the leader. However, two large deletions that blocked translation could be reverted to activator dependent expression by sec ondary changes in the remaining 5'-UTL sequences, indicating that the original deletions had not removed the translational activator target but only deformed it. Taken together, the deletion mutations and rever tants define a region of 151 bases (between positions -480 and -330 re lative to the start codon) containing sequences that are sufficient fo r translational activation when modified slightly. Suppression of the respiratory phenotypes of two 5'-UTL mutations by overexpression of PE T54, PET122, and PET494 indicated functional interactions between the leader and the three activator proteins, The mature COX3 mRNA is cleav ed from a precursor immediately downstream of the preceding tRNA(Val) in a fashion resembling mRNA processing in vertebrate mitochondria, Ou r results indicate that the site of this cleavage in Saccharomyces cer evisiae is determined solely by the position of the tRNA.