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

Abstract

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 (A₇₃). 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 A₇₃, 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 A₇₃ in tRNA^His revealed that the G₋₁ addition reaction was more efficient onto the template containing the opposite A₇₃ than onto the template with cytidine (C₇₃) or other bases forming canonical Watson–Crick base-pairing. Some interaction might occur between incoming GTP and A₇₃, 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.