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

摘要

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 ofd-/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 tRNATyrA76 in the catalysis by TyrRS and DTD remained unknown. To address this issue, [32P]-labeled tRNATyrsubstrates have been tested in aminoacylation and deacylation assays. TyrRS demonstrates similar activity in charging the 2′ and 3′-OH groups of A76 withl-Tyr. This synthetase can effectively use both OH groups as primary sites for aminoacylation withl-Tyr, but demonstrates severe preference toward 2′-OH, in charging withd-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 ofd-Tyr-tRNATyrspecifically from the 3′-OH group, while the 2′-OH assists in this hydrolysis.