Interpretation of tRNA-mischarging kinetics

Abstract

Incorrect tRNA aminoacylation reactions are characterized by very slow reaction rates and by the fact that in most cases they are incomplete. In a previous study some of us explained the incompleteness of the correct aminoacylation reactions of tRNA, which can be encountered under certain experimental conditions (for instance low enzyme concentration or high ionic strength) by an equilibrium between the aminoacylation and the deacylation reactions [J. Bonnet and J.P. Ebel (1972) Eur. J.Biochem. 31, 335-344]. In the present report we bring evidence that the incorrect valylation of yeast tRNAfMet by yeast valyl-tRNA synthetase studied under standard experimental conditions, can also be described by a kinetic rate law including the rate equations of the aminoacylation and of the various deacylation reactions. In particular we show that the incomplete mischarging plateaus reflect the existence of an equilibrium between the valylation reaction on the one hand and the spontaneous and enzymic deacylation of valyl-tRNAfMet and the reverse of the valylation reaction on the other hand. However, when the valyl-tRNA synthetase concentration is not very high the reverse reaction of the amino-acylation does not play a predominant part in the establishment of the plateau. These interpretations have been extended to other mischarging systems: valylation of yeast tRNAPhE by yeast valyl-tRNA synthetase and mischarging of tRNAfMet and tRNA2Val from yeast by yeast phenylalanyl-tRNA synthetase. Unusual mischarging kinetics have been discussed. Furthermore, and as in correct systems, we found that during the mischarging of tRNAfMet one ATP is hydrolyzed per tRNA charged with valine. We conclude that the correct and the incorrect amino-acylation of tRNA behave kinetically in a similar way.