Human Thg1 displays tRNA-inducible GTPase activity

tRNA^His guanylyltransferase (Thg1) catalyzes the 3^'-5^' incorporation of guanosine into position -1 (G-1) of tRNA^His. G-1 is unique to tRNA^His and is crucial for recognition by histidyl-tRNA synthetase (HisRS). Yeast Thg1 requires ATP for G-1 addition to tRNA^His opposite A73, whereas archaeal Thg1 requires either ATP or GTP for G-1 addition to tRNA^His opposite C73. Paradoxically, human Thg1 (HsThg1) can add G-1 to tRNAs^His with A73 (cytoplasmic) and C73 (mitochondrial). As N73 is immediately followed by a CCA end (positions 74–76), how HsThg1 prevents successive 3^'-5^' incorporation of G-1/G-2/G-3 into mitochondrial tRNA^His (tRNA_m^His) through a template-dependent mechanism remains a puzzle. We showed herein that mature native human tRNA_m^His indeed contains only G-1. ATP was absolutely required for G-1 addition to tRNA_m^His by HsThg1. Although HsThg1 could incorporate more than one GTP into tRNA_m^Hisin vitro, a single-GTP incorporation prevailed when the relative GTP level was low. Surprisingly, HsThg1 possessed a tRNA-inducible GTPase activity, which could be inhibited by ATP. Similar activity was found in other high-eukaryotic dual-functional Thg1 enzymes, but not in yeast Thg1.