| dc.description.abstract | Abstract
So far, only the GRS1 gene has been reported to use a naturally occurring non-AUG triplet as the translation initiator in Saccharomyces cerevisiae. Recently, our lab has discovered a second example of non-AUG initiation in the yeast. ALA1 is the only gene in Saccharomyces cerevisiae (alanyl-tRNA synthetase, AlaRS), encodes both cytoplasmic and mitochondrial forms of the enzyme. The former is translationally initiated at the AUG codon (designated as AUG1) close to the 5’-end of its open reading frame, while the latter is initiated from upstream in-frame redundant non-AUG codons (i.e., ACG-25 and ACG-24). In this thesis, I investigated the transcriptional and translational profiles of ALA1, aiming at elucidating the mechanism that is responsible for the bifunctional phenotype of this gene. Transcriptional mapping reveals the existence of three overlapping transcripts for ALA1, with 5’ ends at positions 117, 105, and 54, respectively, relative to the “A” nucleotide of AUG1. Western blot analysis further confirms the initiating activity of the redundant ACG codons. The frequency of initiation at ACG-25/ACG-24 is around 30% relative to that at AUG-25. Results also suggest that certain non-AUG codons in the presequence, such as UUG-16 and AUA-1, are also involved in the synthesis of minor protein isoforms. Unexpectedly, mutations that destroyed the pseudoknot structure, downstream of the redundant ACG codons, did not impair this initiating activity. Taken together, our results suggest that non-AUG initiation is a mechanism more widespread than previously thought. | en_US |