Recognition of tRNA^His in an RNase P-Free Nanoarchaeum

The 5' extra guanosine with 5'-monophosphate at position -1 (G-1) of tRNA^His (p-tRNA^His) is a nearly universal feature that establishes tRNA^His identity. G-1 is either genome encoded and retained after processing by RNase P (RNase P) or posttranscriptionally incorporated by tRNA^His guanylyltransferase (Thg1) after RNase P cleavage. However, RNase P is not found in the hyperthermophilic archaeum Nanoarchaeum equitans; instead, all of its tRNAs, including tRNA^His, are transcribed as leaderless tRNAs with 5'-triphosphate (ppp-tRNAs). How N. equitans histidyl-tRNA synthetase (NeHisRS) recognizes its cognate tRNA (NetRNA^His) is of particular interest. In this paper, we show that G-1 serves as the major identity element of NetRNA^His, with its anticodon performing a similar role, though to a lesser extent. Moreover, NeHisRS distinctly preferred p-tRNA^His over ppp-tRNA^His (~5-fold difference). Unlike other prokaryotic HisRSs, which strongly prefer tRNA^His with C73, this enzyme could charge tRNAs^His with A73 and C73 with nearly equal efficiency. As a result, mutation at the C73-recognition amino acid residue Q112 had only a minor effect (<2-fold reduction). This study suggests that NeHisRS has evolved to disregard C73, but it still maintains its evolutionarily preserved preference toward tRNA^His with 5'-monophosphate. IMPORTANCE Mature tRNA^His has, at its 5'-terminus, an extra guanosine with 5'-monophosphate, designated G-1. G-1 is the major recognition element for histidyl-tRNA synthetase (HisRS), regardless of whether it is of eukaryotic or prokaryotic origin. However, in the hyperthermophilic archaeum Nanoarchaeum equitans, all its tRNAs, including tRNA^His, are transcribed as leaderless tRNAs with 5'-triphosphate. This piqued our curiosity about whether N. equitans histidyl-tRNA synthetase (NeHisRS) prefers tRNA^His with 5'-triphosphate. We show herein that G-1 is still the major recognition element for NeHisRS. However, unlike other prokaryotic HisRSs, which strongly prefer tRNA^His with C73, this enzyme shows almost the same preference for C73 and A73. Most intriguingly, NeHisRS still prefers 5'-monophosphate over 5'-triphosphate. It thus appears that the preference of HisRS for tRNA^His with 5'-monophosphate emerged very early in evolution.