Recognition by tryptophanyl-tRNA synthetases of discriminator base on tRNA^Trp from three biological domains

To study the recognition by tryptophanyl-tRNA synthetase (TrpRS) of tRNA^Trp discriminator base, mutations were introduced into the discriminator base of Bacillus subtilis, Archeoglobus fulgidus, and bovine tRNA^Trp, representing the three biological domains. When B. subtilis, A. fulgidus, and human TrpRS were used to acylate these tRNA^Trp, two distinct preference profiles regarding the discriminator base of different tRNA^Trp substrates were found: G>A>U>C for B. subtilis TrpRS, and A>C>U>G for A. fulgidus and human TrpRS. The preference for G73 in tRNA^Trp by bacterial TrpRS is much stronger than the modest preferences for A73 by the archaeal and eukaryotic TrpRS. Cross-species reactivities between TrpRS and tRNA^Trp from the three domains were in accordance with the view that the evolutionary position of archaea is intermediate between those of eukarya and bacteria. NMR spectroscopy revealed that mutation of A73 to G73 in bovine tRNA^Trp elicited a conformational alteration in the G1-C72 base pair. Mutation of G1-C72 to A1-U72 or disruption of the G1-C72 base pair also caused reduction of Trp-tRNA^Trp formation. These observations identify a tRNA^Trp structural region near the end of acceptor stem comprising A73 and G1-C72 as a crucial domain required for effective recognition by human TrpRS.