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

McElroy, Craig Alan

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2005, Doctor of Philosophy, Ohio State University, Ohio State Biochemistry Program.
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). Next we used isothermal titration calorimetry (ITC) and 15N relaxation measurements to gain quantitative insight into the dynamics and thermodynamics of this binding-coupled folding event. By performing titrations of tryptophan into TRAP at temperatures from 25 to 50 °C we have determined a heat capacity change of -0.366 kcal M-1 K-1. Estimation from the crystal structure of the change in solvent accessible surface area between apo- and holo-TRAP approximates that only 87.5 cal M-1 K-1 of this change in heat capacity is accounted for by burial of the tryptophan ligand in the tryptophan-binding site. We have demonstrated that proton and ion linkage effects do not contribute significantly to the change in heat capacity, and therefore conclude that the excess change in heat capacity is due to binding-coupled protein folding of 17.2-19.9 residues per monomer of TRAP. 15N relaxation measurements and hydrogen/deuterium (H/D) exchange were used to determine the time-scale of the motions that are occurring in apo-TRAP and the change in these motions upon tryptophan binding. The relaxation measurements and model-free analysis show similar motional amplitudes on the ps-ns timescale in apo and tryptophan-bound TRAP, but large differences in the exchange terms reflecting ms-ms timescale motions. H/D exchange measurements demonstrate that both apo and tryptophan-bound TRAP contain regions of the protein that are largely unprotected from the solvent as well as regions of the protein that are almost completely protected from the solvent. These studies demonstrate that ~20 residues fold into place and the dynamics of TRAP on the ms-ms timescale change upon tryptophan binding, which is consistent with the previously mentioned allosteric model. Finally, we have studied the oligomeric state of B. subtilis AT using NMR translational and rotational diffusion analysis, dynamic light scattering, and native ESI mass spectrometry and found that AT is present as a homododecamer in solution. We have also used NMR to make backbone resonance assignments of the 68 kDa AT oligomer. 15N relaxation measurements and model-free analysis were used to determine the dynamics of AT, showing motions present on both the ps-ns timescale as well as exchange on the ms-ms timescale. Finally, NMR studies of the TRAP-AT interaction were also performed, revealing that all of the resonances of TRAP disappear upon its interaction with AT, whereas only some of the resonances of AT seem to be affected, consistent with loss of molecular symmetry upon TRAP-AT binding.
Mark Foster (Advisor)
343 p.

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Citations

  • McElroy, C. A. (2005). Dynamics and function: mechanistic studies of the gene regulatory proteins TRAP and anti-TRAP [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1111429539

    APA Style (7th edition)

  • McElroy, Craig. Dynamics and function: mechanistic studies of the gene regulatory proteins TRAP and anti-TRAP. 2005. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1111429539.

    MLA Style (8th edition)

  • McElroy, Craig. "Dynamics and function: mechanistic studies of the gene regulatory proteins TRAP and anti-TRAP." Doctoral dissertation, Ohio State University, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=osu1111429539

    Chicago Manual of Style (17th edition)