Analysis of the TRAP-frp leader RNA interaction

In Bacillus subtilis, expression of the tryptophafl biosynthetic genes is regulated in response to tryptophan by an RNA binding protein called TRAP (trp RNA binding Attenuation Prolein). TRAP, which has been recently shown to exist as an elevenmer of identical subunits arranged in a symmetrical ring, binds RNA at a series of evenly spaced GAG or I.'AG repeats in both the leader region of the trp operon and the 5 untranslated region of trpG. Based on the structure of TRAP and its RNA binding sites, we have proposed that RNA binds TRAP by wrapping around the protein with each G/UAG repeal making contacts with one or a combination of two adjacent subunits. Furthermore, TRAP only binds its RNA target when activated by binding eleven molecules of tryptophan- Due to the unusual nature of the proposed TRAP:RNA interaction, we have begun characterize this interaction. We have shown that TRAP binds trp leader RNA with a Kd-0.12 nM in the presence of Im M L-trp. We have also shown that the AH of the TRAP:RNA interaction is highly unfavorable at +15.9 kcai mol-1 and is compensated by a AS of +97 cal mol-1 K-l. The rate of RNA dissociation from the RNA:TRAP:tryptophan ternary complex was found to be very slow in high concentrations of tryptophan (>4U uM) hut increased as the concentration of tryptophan was lowered. This suggests that dissociation of tryptophan from the ternary complex is the rate limiting step in RNA dissociation. Finally, results from both glycero) gradients and mobility shift gels suggest that two TRAP eievnemers bind to each trp leaderRN'A and this is currently under investigation.