The Dual Role of the 2'-OH Group of A76 tRNATyr in the Prevention of d-tyrosine Mistranslation.

ABSTRACT

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 tRNATyr A76 in the catalysis by TyrRS and DTD remained unknown. To address this issue, [32P]-labeled tRNATyr 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-tRNATyr specifically from the 3'-OH group, while the 2'-OH assists in this hydrolysis.