Abstract
AbstractLittle is known about the thermodynamic forces that drive the folding pathways of higher-order RNA structure. In this study, we employ calorimetric [isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC)] and spectroscopic (NMR and UV) methods to characterize the thermodynamics of the GAAA tetraloop–receptor interaction, utilizing a previously described bivalent construct. ITC studies indicate that the bivalent interaction is enthalpy driven and highly stable, with a binding constant (Kobs) of 5.5×106 M−1 and enthalpy (ΔHobso) of −33.8 kcal/mol at 45 °C in 20 mM KCl and 2 mM MgCl2. Thus, we derive the ΔHobso for a single tetraloop–receptor interaction to be −16.9 kcal/mol at these conditions. UV absorbance data indicate that an increase in base stacking quality contributes to the enthalpy of complex formation. These highly favorable thermodynamics are consistent with the known critical role for the tetraloop–receptor motif in the folding of large RNAs. Additionally, a significant heat capacity change (ΔCp,obso) of −0.24 kcal mol−1 K−1 was determined by ITC. DSC and UV-monitored thermal denaturation experiments indicate that the bivalent tetraloop–receptor construct follows a minimally five-state unfolding pathway and suggest the observed ΔCp,obso for the interaction results from a temperature-dependent unbound receptor RNA structure.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have