In Bacillus subtilis, expression of the tryptophan biosynthetic (trp) genes is negatively regulated in response to the availability of tryptophan by a 75 amino acid RNA-binding protein called TRAP. TRAP regulates both transcription and translation of these genes by binding to specific RNA target sites in a tryptophan dependent manner. In the presence of excess tryptophan, the TRAP-Trp complex is activated to bind a specific target in the trp leader transcript. This binding induces formation of a transcription terminator and transcription halts in the leader region. In the absence of tryptophan, apo-TRAP does not bind RNA, and the anti-terminator forms and the operon is expressed. The structure of TRAP in complex with L-tryptophan has been solved by X-ray crystallography, and reveals a novel arrangement, in which eleven identical subunits are arranged in a ring structure with a 23 - central hole. TRAP is the first protein described that contains 11 identical subunits. The TRAP-Trp oligomer is composed of eleven 7-stranded antiparallel b-sheets, each containing 4 b-strands from one subunit and 3 strands from the adjacent subunit. The goal of the proposed studies is to provide a structural and dynamical basis for understanding the mechanism by which TRAP regulates expression of the tryptophan biosynthetic genes in response to tryptophan availability. To accomplish this goal, one must understand how tryptophan binds to the apo-protein, and how the binding of Trp activates the TRAP protein to bind its RNA target. Extensive efforts to generate suitable crystals of apo-TRAP have been unsuccessful, and current prospects for an X-ray based structure of the apo-state are not encouraging. To test the Trp-binding hypothesis, and to further probe the structure-function relationship in the TRAP-activated system, the investigators intend to use high resolution multidimensional multinuclear NMR techniques, coupled with distance geometry/simulated annealing procedures, to determine the structure and dynamics of the TRAP protein in the absence and presence of tryptophan, and in a ternary complex with both Trp and RNA. This proposal presents results that confirm the feasibility of the NMR-based approach.
