| Summary: | 博士 === 國立中央大學 === 生命科學系 === 107 === The extra 5’ guanine nucleotide (G-1) on tRNAHis is a nearly universal feature that specifies tRNAHis identity. The G-1 residue is either genome encoded or post-transcriptionally added by tRNAHis guanylyltransferase (Thg1). Despite Caenorhabditis elegans being a Thg1-independent organism, its cytoplasmic tRNAHis retains a genome-encoded G-1. Our study showed that its mitochondrial tRNAHis lacks G-1. This tRNA, while lacking the canonical identity element, can still be efficiently aminoacylated in vivo. Mutagenesis assays showed that the anticodon takes a primary role in C. elegans tRNAHis identity recognition, being comparable to the universal identity element. On the other hand, the discriminator base N73 is a key identity element of tRNAHis. Humans possess two distinct yet closely related histidyl-tRNA synthetase (HisRS) homologues, despite these two isoforms sharing high sequence similarities (81% identity), they strongly preferred different discriminator bases (A73 or C73). Most intriguingly, swapping the discriminator base between the cytoplasmic and mitochondrial tRNAHis isoacceptors conveniently switched their enzyme preferences. Moreover, humans possess a single Thg1 gene is responsible for modification of both the cytoplasmic (with A73) and mitochondrial (with C73) tRNAHis isoacceptors. We reported herein that human cytoplasmic tRNAHis was modified via an ATP-dependent mechanism, while its mitochondrial tRNA isoacceptor was modified via a GTP-dependent mechanism. While N73 affected the efficiency of G-1 addition by Thg1, mutation of N73 (A73 to C or C73 to A) did not switch the mechanisms used for modification. Most intriguingly, the GTP-dependent mechanism was inhibited by ATP. Our study reveals novel scenarios of recognition and modification of tRNAHis in higher eukaryotes.
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