Abstract
Although initiation of translation at non-AUG codons occurs occasionally in prokaryotes and higher eukaryotes, it has not been reported in yeast until very recently. Evidence presented here shows that redundant ACG codons are recognized as alternative translation start sites for ALA1, the only gene in Saccharomyces cerevisiae coding for alanyl-tRNA synthetase. ALA1 is shown to be a bifunctional gene that provides both cytoplasmic and mitochondrial activities. Unlike most bifunctional genes that contain alternative in-frame AUG initiators, there is only one AUG codon, designated AUG1, close to the 5'-end of the ALA1 open reading frame. Transcriptional mapping identified three overlapping transcripts, with 5'-ends at positions 54, 105, and 117 nucleotides upstream of AUG1, respectively. Site-specific mutagenesis demonstrated that the cytoplasmic and mitochondrial functions of ALA1 are provided by two protein isoforms with distinct amino termini; that is, a short cytoplasmic form initiated at AUG1 and a longer mitochondrial isoform initiated at two upstream in-frame ACG codons, i.e. ACG(-25) and ACG(-24). These two ACG codons function redundantly in initiation of translation. Either codon can function in the absence of the other. The short transcript appears to serve as the template for the cytoplasmic form, whereas the longer transcripts are likely to code for both isoforms via alternative initiation. Because yeast ribosomes in general cannot efficiently recognize a non-AUG initiator, this unique feature of redundancy of non-AUG initiators in a single mRNA may in itself represent a novel paradigm for translation initiation from poor initiators.
Highlights
Initiation of translation at non-AUG codons occurs occasionally in prokaryotes and higher eukaryotes, it has not been reported in yeast until very recently
Site-specific mutagenesis demonstrated that the cytoplasmic and mitochondrial functions of ALA1 are provided by two protein isoforms with distinct amino termini; that is, a short cytoplasmic form initiated at AUG1 and a longer mitochondrial isoform initiated at two upstream in-frame ACG codons, i.e. ACG؊25 and ACG؊24
Because the Arabidopsis gene for alanyl-tRNA synthetase (AlaRS) codes for both the cytoplasmic and mitochondrial forms through alternative use of two in-frame ATG codons [22], we decided to test if yeast instead use a non-ATG codon to initiate the translation of its mitochondrial form of AlaRS
Summary
AlaRS, alanyl-tRNA synthetase; ADH, alcohol dehydrogenase; 5-FOA, 5-fluoroorotic acid; RACE, rapid amplification of cDNA ends; YPG, yeast extract peptone glycerol; RT, reverse transcription. Only two yeast genes, GRS1 (one of the two glycyl-tRNA synthetase genes in S. cerevisiae) [16] and CARP2A (the only gene coding for acidic ribosomal protein P2A in Candida albicans) [17], have been reported to use naturally occurring non-AUG triplets as translation initiators. A short form that is responsible for the cytoplasmic activity is translationally initiated from a classical AUG initiator, whereas a longer isoform that is responsible for the mitochondrial activity is initiated from an upstream inframe UUG codon [16]. These two isoforms function exclusively in their respective compartments and, cannot substitute for each other under normal conditions. Considering the fact that yeast ribosomes initiate poorly from a non-AUG triplet, we surmised that the second ACG triplet might serve as a remedial initiation site for the mitochondrial form in case of initiation failure at the first ACG
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