Principles of tRNA^Ala Selection by Alanyl-tRNA Synthetase Based on the Critical G3·U70 Base Pair

Throughout evolution, the presence of a single G3·U70 mismatch in the acceptor stem of tRNA^Ala is the major determinant for aminoacylation with alanine by alanyl-tRNA synthetase (AlaRS). Recently reported crystal structures of the complexes AlaRS-tRNA^Ala/G3·U70 and AlaRS-tRNA^Ala/A3·U70 suggest two very different conformations, representing a reactive and a nonreactive state, respectively. On the basis of these structures, it has been proposed that the G3·U70 base pair guides the -CCA end of the tRNA acceptor stem into the active site of AlaRS, thereby enabling aminoacylation. The crystal structures open up the possibility of directly computing the energetics of tRNA specificity by AlaRS. We have carried out molecular dynamics free-energy simulations to quantitatively estimate tRNA discrimination by AlaRS, focusing on the mutations of the single critical base pair G3·U70 to uncover the energetics underlying the accuracy of tRNA selection. The calculations show that the reactive complex is highly selective in favor of the cognate tRNA^Ala/G3·U70 over its noncognate analogues (A3·U70/G3·C70/A3·C70). In contrast, the nonreactive complex is predicted to be unselective between tRNA^Ala/G3·U70 and tRNA^Ala/A3·U70. Utilizing our calculated relative binding free energies, we show how a simple three-step kinetic scheme for aminoacylation, involving both an initial nonspecific binding step and a subsequent transition to a selective reactive complex, accounts for the observed kinetics of the process.