Transfer RNA identity contributes to transition state stabilization during aminoacyl-tRNA synthesis

Sequence-specific interactions between aminoacyl-tRNA synthetases and their cognate tRNAs ensure both accurate RNA recognition and the efficient catalysis of aminoacylation. The effects of tRNA(Trp) variants on the aminoacylation reaction catalyzed by wild-type Escherichia coli tryptophanyl-tRNA synthetase (TrpRS) have now been investigated by stopped-flow fluorimetry, which allowed a pre-steady-state analysis to be undertaken. This showed that tRNA(Trp) identity has some effect on the ability of tRNA to bind the reaction intermediate TrpRS-tryptophanyl adenylate, but predominantly affects the rate at which tryptophan is transferred from TrpRS-tryptophanyl adenylate to tRNA. Use of the binding (K(tRNA)) and rate constants (k₄) to determine the energetic levels of the various species in the aminoacylation reaction showed a difference of ~ 2 kcal mol^-1 in the barrier to transition state formation compared to wild-type for both tRNA(Trp) A→C73 and tRNA(Gln)CCA. These results directly show that tRNA identity contributes to the degree of complementarity to the transition state for tRNA charging in the active site of an aminoacyl-tRNA synthetase:aminoacyl-adenylate:tRNA complex.