Investigating the Conformational Dynamics of RNA Induced by Metal Cations and Aminoglycosides using NMR

[摘要] Many regulatory RNAs adaptively change their conformation on binding to cognate protein and ligand targets or therapeutic molecules but the mechanism by which these conformational changes occur still remains poorly understood. In this thesis we characterize the dynamic properties of two RNA drug targets; HIV-1 TAR RNA and ribosomal A-site rRNA using a battery of NMR experiments that provide information on motions occurring over picosecond to millisecond timescales. We have used residual dipolar couplings (RDCs) in concert with structure-based electrostatic calculations to characterize the dependence of local and inter-helical recognition motions in TAR containing a trinucleotide pyrimidine bulge on the concentration of Na+ and Mg2+. Results revealed that Na+ or Mg2+ induce a similardynamic transition of TAR from an electrostatic relaxed bent and flexible state to a globally rigid coaxial state, which has a stronger negative charge density and association with counterions. The dynamic transition carries the TAR structure through several of the ligand bound conformations, indicating that metals and electrostatic interactions likely play an important role in adaptive recognition. We used domain-elongation spin relaxation and relaxation dispersion NMR experiments to characterize base flipping and other local motions in the A-site rRNA in the absence and presence of the aminoglycoside antibiotic paromomycin. Our results strongly suggest that A-site can dynamically access conformations in which the two adenines are flipped out at microsecond timescales and that binding to paromomycin is not necessary to induce this transition. These results were then compared to those obtained on a corresponding construct bearing an antibiotic resistance A1408G mutation. This single mutation leads to dramatic changes in the dynamics observed over picosecond to millisecond timescales. We propose that a G-A base-pair reduces the propensity to have both adenine residues looped out, thereby explaining in part the much lower antibiotic affinity for this RNA construct. Taken together, our results provide fundamental new insights into how internal motions occurring on different timescales can drive the conformational changes that accompany molecular recognition.

[发布机构] University of Michigan

[效力级别] A-site RRNA

[关键词] HIV-1 TAR RNA;A-site RRNA;Residual Dipolar Couplings;Carbon Relaxation Dispersion;Metal Ions;Biological Chemistry;Science;Chemistry