Molecular mechanism of substrate recognition and specificity of tRNA(His) guanylyltransferase during nucleotide addition in the 3′–5′ direction

The tRNA(His) guanylyltransferase (Thg1) transfers a guanosine triphosphate (GTP) in the 3′–5′ direction onto the 5′-terminal of tRNA(His), opposite adenosine at position 73 (A(73)). The guanosine at the −1 position (G(−1)) serves as an identity element for histidyl-tRNA synthetase. To investigate the mechanism of recognition for the insertion of GTP opposite A(73), first we constructed a two-stranded tRNA(His) molecule composed of a primer and a template strand through division at the D-loop. Next, we evaluated the structural requirements of the incoming GTP from the incorporation efficiencies of GTP analogs into the two-piece tRNA(His). Nitrogen at position 7 and the 6-keto oxygen of the guanine base were important for G(−1) addition; however, interestingly, the 2-amino group was found not to be essential from the highest incorporation efficiency of inosine triphosphate. Furthermore, substitution of the conserved A(73) in tRNA(His) revealed that the G(−1) addition reaction was more efficient onto the template containing the opposite A(73) than onto the template with cytidine (C(73)) or other bases forming canonical Watson–Crick base-pairing. Some interaction might occur between incoming GTP and A(73), which plays a role in the prevention of continuous templated 3′–5′ polymerization. This study provides important insights into the mechanism of accurate tRNA(His) maturation.