Ligation of tRNAs with their cognate amino acids, by aminoacyl-tRNA synthetases, establishes the genetic code. Throughout evolution, tRNA(Ala) selection by alanyl-tRNAsynthetase (AlaRS) has depended predominantly on a single wobble base pair in the acceptor stem, G3 center dot U70, mainly on the k(cat) level. Here we report the crystal structures of an archaeal AlaRS in complex with tRNAAla with G3 center dot U70 and its A3 center dot U70 variant. AlaRS interacts with both the minor-and the major-groove sides of G3 center dot U70, widening the major groove. The geometry difference between G3 center dot U70 and A3 center dot U70 is transmitted along the acceptor stemto the 3'-CCA region. Thus, the 3'-CCA region of tRNA(Ala) with G3 center dot U70 is oriented to the reactive route that reaches the active site, whereas that of the A3 center dot U70 variant is folded back into the non-reactive route. This novel mechanism enables the single wobble pair to dominantly determine the specificity of tRNA selection, by an approximate 100-fold difference in k(cat) .