Recognition of tRNAHis in an RNase P-Free Nanoarchaeum

The 59 extra guanosine with 59-monophosphate at position-1 (G-1) of tRNAHis (p-TRNAHis) is a nearly universal feature that establishes tRNAHis identity. G-1 is either genome encoded and retained after processing by RNase P (RNase P) or posttranscriptionally incorporated by tRNAHis guanylyltransferase (Thg1) after RNase P cleavage. However, RNase P is not found in the hyperthermophilic archaeum Nanoarchaeum equitans; instead, all of its tRNAs, including tRNAHis, are transcribed as leaderless tRNAs with 59-Triphosphate (ppptRNAs). How N. equitans histidyl-TRNA synthetase (NeHisRS) recognizes its cognate tRNA (NetRNAHis) is of particular interest. In this paper, we show that G-1 serves as the major identity element of NetRNAHis, with its anticodon performing a similar role, though to a lesser extent. Moreover, NeHisRS distinctly preferred p-TRNAHis over ppp-TRNAHis (;5-fold difference). Unlike other prokaryotic HisRSs, which strongly prefer tRNAHis with C73, this enzyme could charge tRNAsHis 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 tRNAHis with 59-monophosphate. IMPORTANCE Mature tRNAHis has, at its 59-Terminus, an extra guanosine with 59-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 tRNAHis, are transcribed as leaderless tRNAs with 59-Triphosphate. This piqued our curiosity about whether N. equitans histidyl-TRNA synthetase (NeHisRS) prefers tRNAHis with 59-Triphosphate. We show herein that G-1 is still the major recognition element for NeHisRS. However, unlike other prokaryotic HisRSs, which strongly prefer tRNAHis with C73, this enzyme shows almost the same preference for C73 and A73. Most intriguingly, NeHisRS still prefers 59-monophosphate over 59-Triphosphate. It thus appears that the preference of HisRS for tRNAHis with 59-monophosphate emerged very early in evolution.