Molecular mechanism of substrate recognition and specificity of tRNA guanylyltransferase during nucleotide addition in the 3'-5' direction.

The tRNA guanylyltransferase (Thg1) transfers a guanosine triphosphate (GTP) in the 3'-5' direction onto the 5'-terminal of tRNA, opposite adenosine at position 73 (A). The guanosine at the -1 position (G) serves as an identity element for histidyl-tRNA synthetase. To investigate the mechanism of recognition for the insertion of GTP opposite A, first we constructed a two-stranded tRNA 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 Nitrogen at position 7 and the 6-keto oxygen of the guanine base were important for G 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 in tRNA revealed that the G addition reaction was more efficient onto the template containing the opposite A than onto the template with cytidine (C) or other bases forming canonical Watson-Crick base-pairing. Some interaction might occur between incoming GTP and A, which plays a role in the prevention of continuous templated 3'-5' polymerization. This study provides important insights into the mechanism of accurate tRNA maturation.