The AUG start codon of the Saccharomyces cerevisiae NFS1 gene can be substituted for by UUG without increased initiation of translation at downstreamcodons

The selection of the site for initiation of translation for the Saccharomyc es cerevisiae NFS1 gene was examined using mutated AUG1, AUG2 and AUG3 codo ns. When AUG1 of the yeast NFS1 gene was mutated to UUG and the resulting m RNA was translated in vitro using a reticulocyte system, initiation from th e mutated codon was abolished and occurred instead at downstream codons at increased rates. When the same mRNA was translated using a yeast extract, t ranslation initiated at the mutated codon, albeit at a reduced rate, and th ere was no increased translation at downstream AUG codons. The NFS1 gene in which AUG1 was replaced by UUG was also able to substitute for the wild-ty pe gene in vivo in yeast Western blots confirmed that the encoded protein w as the same size as that encoded by the wild-type gene and that both the wi ld-type and mutated proteins localized to mitochondria. This is apparently the first example of a yeast protein where mutagenesis of AUG1 does not lea d to alternate use of a downstream AUG.