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.