Conformational Flexibility and Dynamics of the Internal Loop and Helical Regions of the Kink-Turn Motif in the Glycine Riboswitch by Site-Directed Spin-Labeling

Site-directed spin-labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy provides a means for a solution state description of site-specific dynamics and flexibility of large RNAs, facilitating our understanding of the effects of environmental conditions such as ligands and ions on RNA structure and dynamics. Here, the utility and capability of EPR line shape analysis and distance measurements to monitor and describe site-specific changes in the conformational dynamics of internal loop nucleobases as well as helix helix interactions of the kink turn motif in the Vibrio cholerae (VC) glycine riboswitch that occur upon sequential K+, Mg2+-, and glycine-induced folding were explored. Spin-labels were incorporated into the 232-nucleotide sequence via splinted ligation strategies. Thiouridine nucleobase labeling within the internal loop reveals unambiguous differential dynamics for two successive sites labeled, with varied rates of motion reflective of base flipping and base stacking. EPR-based distance measurements for nitroxide spin-labels incorporated within the RNA backbone in the helical regions of the kink turn motif are reflective of helical formation and tertiary interaction induced by ion stabilization. In both instances, results indicate that the structural formation of the kink turn motif in the VC glycine riboswitch can be stabilized by 100 mM K+ where the conformational flexibility of the kink turn motif is not further tightened by subsequent addition of divalent ions. Although glycine binding is likely to induce structural and dynamic changes in other regions, SDSL indicates no impact of glycine binding on the local dynamics or structure of the kink turn motif as investigated here. Overall, these results demonstrate the ability of SDSL to interrogate site-specific base dynamics and packing of helices in large RNAs and demonstrate ion-induced stability of the kink turn fold of the VC riboswitch.