The Dual Role of the 2′-OH Group of A76 tRNA^Tyr in the Prevention of D-tyrosine Mistranslation

Aminoacyl-tRNA-synthetases are crucial enzymes for initiation step of translation. Possessing editing activity, they protect living cells from misincorporation of non-cognate and non-proteinogenic amino acids into proteins. Tyrosyl-tRNA synthetase (TyrRS) does not have such editing properties, but it shares weak stereospecificity in recognition of D-/L-tyrosine (Tyr). Nevertheless, an additional enzyme, D-aminoacyl-tRNA-deacylase (DTD), exists to overcome these deficiencies. The precise catalytic role of hydroxyl groups of the tRNA^Tyr A76 in the catalysis by TyrRS and DTD remained unknown. To address this issue, [³²P]-labeled tRNA^Tyr substrates have been tested in aminoacylation and deacylation assays. TyrRS demonstrates similar activity in charging the 2′ and 3′-OH groups of A76 with L-Tyr. This synthetase can effectively use both OH groups as primary sites for aminoacylation with L-Tyr, but demonstrates severe preference toward 2′-OH, in charging with D-Tyr. In both cases, the catalysis is not substrate-assisted: neither the 2′-OH nor the 3′-OH group assists catalysis. In contrast, DTD catalyzes deacylation of D-Tyr-tRNA^Tyr specifically from the 3′-OH group, while the 2′-OH assists in this hydrolysis.