Dynamics and function: Mechanistic studies of the gene regulatory proteins TRAP and anti-TRAP

In most Bacilli the trp RNA-binding attenuation protein (TRAP) is responsible for responding to the intracellular levels of tryptophan and coordinately regulating the transcription and in many cases the translation of the genes required for tryptophan metabolism. Both transcriptional and translational control by TRAP depend on the ability of tryptophan-activated TRAP to bind to an RNA site composed of multiple G/UAG repeats that are generally separated by two or three nonconserved spacer nucleotides. Crystal structures of TRAP in complex with tryptophan and in ternary complex with tryptophan and RNA are available. However, no structure is available of TRAP in the absence of tryptophan; thus, the mechanism of allostery remains unclear. Not only are these genes regulated in response to tryptophan levels, but the levels of uncharged tRNATrp are also sensed through the T-box antitermination mechanism, which regulates the transcription of the yczA gene, or anti-TRAP (AT). When uncharged tRNATrp accumulates, transcription of the gene encoding AT is activated. Once translated, AT binds to tryptophan-activated TRAP, preventing RNA-binding and thereby abrogating TRAP-mediated regulation of gene expression.; We have used TROSY-based NMR experiments to study the mechanism of ligand-mediated allosteric regulation in the 90.6 kDa (11-mer) TRAP. By recording a series of two-dimensional 15N-edited TROSY NMR spectra of TRAP from the thermophile Bacillus stearothermophilus over an extended range of temperatures, we have found tryptophan binding to be temperature dependent in agreement with the previously observed temperature dependent RNA binding. Triple-resonance TROSY-based NMR spectra recorded at 55°C have allowed us to obtain backbone resonance assignments for uniformly 2H/13C/15N-labeled TRAP both in the inactive and active forms (free and bound to tryptophan). Based on ligand-dependent differential line broadening and chemical shift perturbations, coupled with the results of proteolytic sensitivity measurements, we infer that tryptophan-modulated protein flexibility (dynamics) plays a central role in TRAP function by altering its RNA binding affinity. Furthermore, because the crystal structures show that the ligand is completely buried in the bound state, we speculate that such dynamic behavior may be important to enable rapid response to changes in intracellular tryptophan levels. Thus we propose a model for the allosteric control of TRAP in which RNA-binding is regulated through a tryptophan-dependent change in protein dynamics (i.e. binding-coupled protein folding). (Abstract shortened by UMI.)...