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.