Naturally occurring dual recognition of tRNA^His substrates with and without a universal identity element

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.