Suppressors of translation initiation defect in hem12 locus of Saccharomyces cerevisiae

A system for the positive selection of transational initiation suppressors in S. cerevisiae has been developed. A mutant with an ATA initiation codon in the HEM12 gene, encoding uroporphyrinogen decarboxylase, was used to sel ect ck and trans-acting suppressors. These suppressors partially restore gr owth on nonfermentable carbon sources, such as glycerol, but still allow th e accumulation of porphyrins. All extragenic suppressors are mapped to the SU11 locus, encoding initiation factor eIF1. The effect of the hem12 mutati on is also partially reversed by the known SUI3 suppressor encoding the bet a subunit of eIF2. In contrast, the sui2 suppressor encoding the a subunit of eIF2 does not affect the hem 12 phenotype. The intragenic suppressors ar e able to restore the translation of hem12 due to the generation of additio nal, in frame AUG codons upstream of the hem12-14 mutation. Mutational analysis of the HEM12 leader sequence was also performed to dete rmine the role of small open reading frames (uORFs) present upstream of the HEM12 ORF. Studies on the expression of integrated hem12-1/4-lacZ fusion, devoid of all upstream ATGs, indicate a lack of regulatory effect of uORFs on HEM12 translation.