Abstract
Previous studies have shown that translation of mrna for yeast glycyl-tRNA synthetase is alternatively initiated from UUG and a downstream AUG initiation codon. Evidence presented here shows that unlike an AUG initiation codon, efficiency of this non-AUG initiation codon is significantly affected by its sequence context, in particular the nucleotides at positions -3 to -1 relative to the initiation codon. A/A/R (R represents A Or G) and C/G/C appear to be the most and least favorable sequences at these positions, respectively. Mutation of the native context sequence -3 to -1 from AAA to CGC reduced translation initiation from the UUG codon up to 32-fold and resulted in loss of mitochondrial respiration. although an AUG initiation codon is, in general, unresponsive to context changes in yeast, an AAA (-3 to -1) to CGC mutation still reduced its initiating activity up to 8-fold under similar conditions. these results suggest that sequence context is more important for translation initiation in yeast than previously appreciated.
Highlights
Aminoacyl-tRNA synthetases are a group of primordial enzymes, each of which catalyzes the attachment of a specific amino acid to its cognate tRNAs
In the case of CARP2A, a non-AUG codon, i.e. GUG, serves as the exclusive translation initiator, whereas in the cases of ALA1 and GRS1, non-AUG codons act as alternative translation initiators that are accompanied by a downstream in-frame AUG initiation codon
The longer form that accounts for the mitochondrial activity is initiated from UUG(Ϫ23) (the number “Ϫ23” in UUG(Ϫ23) refers to the 23rd codon triplet that precedes AUG1), whereas the short form that is responsible for the cytoplasmic activity is initiated from AUG1 (Fig. 1) [11]
Summary
AlaRS, alanyl-tRNA synthetase; ADH, alcohol dehydrogenase; GlyRS, glycyl-tRNA synthetase; YPG, yeast extract-peptone-glycerol; eIF, eukaryotic initiation factor; RT, reverse transcription; -gal, -galactosidase. AUG was relatively insensitive to context changes under similar conditions, an AAA (Ϫ3 to Ϫ1) to CGC mutation still reduced its initiating activity up to 8-fold, suggesting that an AUG initiation codon could be subject to the same rule of modulation, albeit to a lesser extent. In this sense, a non-AUG initiator could perhaps serve as a better model in identifying the sequence and structural elements important for translation initiation in yeast
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