Kinetic and thermodynamic analysis of the interaction between TRAP (trp RNA-binding attenuation protein) of Bacillus subtilis and trp leader RNA

In Bacillus subtilis, expression of the tryptophan biosynthetic genes is regulated in response to tryptophan by an RNA-binding protein called TRAP (trp RNA-binding attenuation protein). TRAP has been shown to contain 11 identical subunits arranged in a symmetrical ring. Kinetic and thermodynamic parameters of the interaction between tryptophan-activated TRAP and trp leader RNA were studied. Results from glycerol gradients and mobility shift gels indicate that two TRAP 11-mers bind to each trp leader RNA. A filter binding assay was used to determine an apparent binding constant of 8.0 ± 1.3 x 10⁹ M^-1 (K(d) = 0.12 ± 0.02 nM) for TRAP and an RNA containing residues +36 to +92 of the trp leader RNA in 1 mM L.tryptophan at 37 °C. The temperature dependence of K(app) was somewhat unexpected demonstrating that the ΔH of the interaction is highly unfavorable at +15.9 kcal mol^-1. Therefore, the interaction is completely driven by a ΔS of +97 cal mol^-1 K^-1. The interaction between tryptophan-activated TRAP and trp leader RNA displayed broad salt and pH activity profiles. Finally, the rate of RNA dissociation from the RNA·TRAP·tryptophan ternary complex was found to be very slow in high concentrations of tryptophan (>40 μM) but increased in lower tryptophan concentrations. This suggests that dissociation of tryptophan from the ternary complex is the rate-limiting step in RNA dissociation.