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 tRNATrP variants on the aminoacylation reaction catalyzed by wild-type Escherichia coli tryptophanyl-tRNA synthe-tase (TrpRS) have now been investigated by stopped-flow fluorimetry, which allowed a pre-steady-state analysis to be undertaken. This showed that tRNATrP identity has some effect on the ability of tRNA to bind the reaction intermediate TrpRS-tryptophanyl-adenylate, but predominantly affects the rate at which trypto-phan is transferred from TrpRS-tryptophanyl ade-nylate to tRNA. Use of the binding (KtRNA) 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 tRNATrP A→C73 an