Anti-Cooperative Binding of Two tRNATyr Molecules to Tyrosyl-tRNA Synthetase from Escherichia coli. Stopped-Flow Investigations Using Unmodified tRNATyr and a Fluorescent Derivative of tRNATyr

Abstract

The kinetics of the interaction of tRNATyr and tyrosyl-tRNA synthetase from Escherichia coli have been investigated by stopped-flow experiments. The association rate constants for the formation of the 1:1 and 2:1 complex as well as the rate constants of the dissociation of these complexes have been determined. The kr-values are 2.2 · 108 and 1.4 · 108 M−1 s−1, the kd-values are 1.5 and 53 s−1, measured in 0.03 M potassium phosphate buffer pH 6. A fluorescent derivative of tRNATyr was prepared by reaction of tRNATyr with fluoresceinisothiocyanate. This labelled tRNA is fully active in the aminoacylation assay; regarding the interaction with the synthetase it shows identical binding parameters as unmodified tRNATyr. Using fluoresceinylthiocarbamoyl-tRNATyr it could be demonstrated that the binding of two tRNA-molecules is anti-cooperative: the binding sites for tRNA on the synthetase are a priori identical, but when one tRNA is bound, the binding of the second tRNA is disfavoured. In the 2:1 complex, however, the tRNAs are again indistinguishable.