Yeast aspartyl-tRNA synthetase residues interacting with tRNA(Asp) identity bases connectively contribute to tRNA(Asp) binding in the ground and transition-state complex and discriminate against non-cognate tRNAs.

Crystallographic studies of the aspartyl-tRNA synthetase-tRNA(Asp)complex from yeast identified on the enzyme a number of residues potentially able to interact with tRNA(Asp). Alanine replacement of these residues (thought to disrupt the interactions) was used in the present study to evaluate their importance in tRNA(Asp)recognition and acylation. The results showed that contacts with the acceptor A of tRNA(Asp)by amino acid residues interacting through their side-chain occur only in the acylation transition state, whereas those located near the G73 discriminator base occur also during initial binding of tRNA(Asp). Interactions with the anticodon bases provide the largest free energy contribution to stability of the enzyme-tRNA complex in its ground state. These contacts also favour catalysis, by acting connectively with each other and with those of G73, as shown by multiple mutant analysis. This implies structural communication transmitting the anticodon recognition signal to the distally located acylation site. This signal might be conveyed via tRNA(Asp)as suggested by the observed conformational change of this molecule upon interaction with AspRS. From binding free energy values corresponding to the different AspRS-tRNA(Asp)interaction domains, it might be concluded that upon complex formation, the anticodon interacts first. Finally, acylation efficiencies of AspRS mutants in the presence of pure tRNA(Asp)and non-fractionated tRNAs indicate that residues involved in the binding of identity bases also discriminate against non-cognate tRNAs.