The fidelity of translation of an mRNA sequence is primarily determined by the accuracy with which the aminoacyl-tRNA synthetases charge their cognate tRNA substrates. The specificity of tRNA recognition is achieved during formation of the transition state complex and involves a conformational change in the enzyme. To elucidate the mechanism of this specific recognition, the following questions will be tested: (1) Do the amino acids which stabilize the pyrophosphate moiety in the transition state complex for the first step of the aminoacylation reaction also stabilize the transition state complex for the second (transfer) step? (2) Do the amino acids which stabilize the second-step transition state complex interact with the 3'-CCA terminus of tRNA(Tyr)? (3) Does the discriminator nucleotide, A73, interact with K151 and/or W196, as well as with active site amino acids? (4) Are the conserved tRNA anticodon loop nucleotides G34, U35, A36 and A38 energetically coupled to one or more of the active site residues that catalyze the tRNA charging reaction? (5) Are conserved polar or charged amino acids in the carboxyl terminal domain of tyrosyl-tRNA synthetase involved in transmission of the signal between the anticodon loop of tRNA(Tyr) and the amino acids at the active site of the synthetase? The first question will be addressed by mutating those amino acids or nucleotide residues believed to play a role in tRNA aminoacylation, and analyzing the effects of these mutations on catalysis. Pre-steady state kinetics will be used in these analyses to quantify the effects of the mutations on the intermediate and transition state complexes. Questions 2 through 5 will be addressed by constructing double mutant free energy cycles to measure the strengths of specific amino acid:nucleotide interactions. Pre-steady state kinetics will be used to assess the effects of the double mutant (e.g., mutated synthetase:mutated tRNA) as well as single mutants and wild-type enzyme/tRNA. By following changes in the strengths of specific interactions between the synthetase and tRNA substrate, the tRNA recognition process can be monitored throughout the course of the reaction.
