A synergistic study of RNA: Utilizing MD simulations and NMR spectroscopy to elucidate the structural dynamics of HIV-1 TAR

Nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations are both powerful methodologies for the characterization of biomolecules. Moreover these techniques are highly complementary, providing the basis for a synergistic approach to the characterization of molecular structure and dynamics. Such a characterization is especially important in the study of ribonucleic acids (RNAs), as the associated conformational dynamics are essential to their function.; In this study a synergistic approach is taken to elucidate the conformational dynamics of the transactivation response (TAR) RNA element of the type-I human immunodeficiency virus (HIV-1). The local and global dynamics of TAR are characterized through NMR obtained and MD computed relaxation parameters, NMR residual dipolar couplings (RDCs), and RDC derived order tensors. These analyses reveal complex multi-timescale dynamics in TAR on both the local and global levels, and reveal that the structural dynamics allow access to the bound states of TAR. Dynamical correlations are also determined, using isotropic reorientational eigenmode dynamics (iRED) analysis, and reveal the hinge residues for the global motions as well as insight into long range correlations throughout the RNA.; In addition to the characterization of they dynamics of TAR, the limits of each technique are explored as well as methods for overcoming such limitations. The affect of structural and experimental errors on the characterization of RNA global dynamics through RDC order tensor analysis when assuming an ideal A-form local structure is explored. Results show that measurement errors and deviations in the assumed structural model do not preclude the ability to accurately determine the global dynamics. A method (Aform-RDC) to estimate the errors in experimentally determined order tensors is presented and tested. Additionally a referencing strategy for the analysis of MD simulations that allows for the quantitative cross-validation of NMR obtained and MD computed dynamical parameters for motionally coupled systems is presented and validated, and using this referencing strategy the timescale limits of NMR relaxation studies and MD simulations are explored.