The discriminator base N73 is a key identity element of tRNA. In eukaryotes, N73 is an "A" in cytoplasmic tRNA and a "C" in mitochondrial tRNA. We present evidence herein that yeast histidyl-tRNA synthetase (HisRS) recognizes both A73 and C73, but somewhat prefers A73 even within the context of mitochondrial tRNA. In contrast, humans possess two distinct yet closely related HisRS homologues, with one encoding the cytoplasmic form (with an extra N-terminal WHEP domain) and the other encoding its mitochondrial counterpart (with an extra N-terminal mitochondrial targeting signal). Despite these two isoforms sharing high sequence similarities (81% identity), they strongly preferred different discriminator bases (A73 or C73). Moreover, only the mitochondrial form recognized the anticodon as a strong identity element. Most intriguingly, swapping the discriminator base between the cytoplasmic and mitochondrial tRNA isoacceptors conveniently switched their enzyme preferences. Similarly, swapping seven residues in the active site between the two isoforms readily switched their N73 preferences. This study suggests that the human HisRS genes, while descending from a common ancestor with dual function for both types of tRNA, have acquired highly specialized tRNA recognition properties through evolution.

The extra 5ʹ guanine nucleotide (G-1) on tRNA His is a nearly universal feature that specifies tRNA His identity. The G-1 residue is either genome encoded or post-transcriptionally added by tRNA His guanylyltransferase (Thg1). Despite Caenorhabditis elegans being a Thg1-independent organism, its cytoplasmic tRNA His (CetRNA n His) retains a genome-encoded G-1. Our study showed that this eukaryote possesses a histidyl-tRNA synthetase (CeHisRS) gene encoding two distinct HisRS isoforms that differ only at their N-termini. Most interestingly, its mitochondrial tRNA His (CetRNA m His ) lacks G-1, a scenario never observed in any organelle. This tRNA, while lacking the canonical identity element, can still be efficiently aminoacylated in vivo. Even so, addition of G-1 to CetRNA m His strongly enhanced its aminoacylation efficiency in vitro. Overexpression of CeHisRS successfully bypassed the requirement for yeast THG1 in the presence of CetRNA n His without G-1. Mutagenesis assays showed that the anticodon takes a primary role in CetRNA His identity recognition, being comparable to the universal identity element. Consequently, simultaneous introduction of both G-1 and the anticodon of tRNA His effectively converted a non-cognate tRNA to a tRNA His -like substrate. Our study suggests that a new balance between identity elements of tRNA His relieves HisRS from the absolute requirement for G-1.

bCytoplasmic and mitochondrial forms of a eukaryotic aminoacyl-tRNA synthetase (aaRS) are generally encoded by two distinct nuclear genes, one of eukaryotic origin and the other of mitochondrial origin. However, in most known yeasts, only the mitochondrial-origin alanyl-tRNA synthetase (AlaRS) gene is retained and plays a dual-functional role. Here, we present a novel scenario of AlaRS evolution in the yeast Vanderwaltozyma polyspora. V. polyspora possesses two significantly diverged AlaRS gene homologues, one encoding the cytoplasmic form and the other its mitochondrial counterpart. Clever selection of transcription and translation initiation sites enables the two isoforms to be localized and thus functional in their respective cellular compartments. However, the two isoforms can also be stably expressed and function in the reciprocal compartments by insertion or removal of a mitochondrial targeting signal. Synteny and phylogeny analyses revealed that the AlaRS homologues of V. polyspora arose from a dual-functional common ancestor through whole-genome duplication (WGD). Moreover, the mitochondrial form had higher synonymous (1.6-fold) and nonsynonymous (2.8-fold) substitution rates than did its cytoplasmic counterpart, presumably due to a lesser constraint imposed on components of the mitochondrial translational apparatus. Our study suggests that asymmetric evolution confers the divergence between the AlaRS paralogues of V. polyspora.