| dc.description.abstract | Abstract
Initiation of protein translation at non-ATG codons has been shown to occur naturally, although rarely in prokaryotes and high eukaryotes, and never in yeast. In this thesis, we provide strong evidence that a non-ATG codon is used as the alternative translational start site of the yeast gene ALA1, which is the only gene in Saccharomyces cerevisiae coding for alanyl-tRNA synthetase. An in vivo functional assay shows that ALA1 is a bifunctional gene that provides both the cytoplasmic and mitochondrial functions. However, unlike most other bifunnctional genes, which contain two alternative in-frame ATG initiators, there is only one ATG codon, designated ATG1, close to the 5’-end of the ALA1open reading frame. Transcriptional mapping reveals the existence of three overlapping transcripts for ALA1, with 5’ ends at positions –143, -105, -54, respectively, relative to the “A” nucleotide of ATG. Site-specific mutagenesis shows that the cytoplasmic and mitochondrial functions of ALA1 are provided by two distinct protein products: a cytoplasmic form initiated at ATG1 and a longer mitochondrial form initiated at two in-frame non-ATG codons, ACG-25 and ACG-24. These two ACG codons function redundantly in initiation of translation. Either one can be functional in the absence of the other; however, the first ACG codon appears to play a predominant role in protein synthesis. Western blot analysis further confirms the initiator function of these two codons. This appears to be the first example in which a non-ATG codon is used physiologically as a transitional initiator in yeast. | en_US |