Thermodynamics and kinetics of RNA tertiary structure formation in the junctionless hairpin ribozyme

Abstract

The hairpin ribozyme consists of two RNA internal loops that interact to form the catalytically active structure. This docking transition is a rare example of intermolecular formation of RNA tertiary structure without coupling to helix annealing. We have used temperature-dependent surface plasmon resonance (SPR) to characterize the thermodynamics and kinetics of RNA tertiary structure formation for the junctionless form of the ribozyme, in which loops A and B reside on separate molecules. We find docking to be strongly enthalpy-driven and to be accompanied by substantial activation barriers for association and dissociation, consistent with the structural reorganization of both internal loops upon complex formation. Comparisons with the parallel analysis of a ribozyme variant carrying a 2′-O-methyl modification at the self-cleavage site and with published data in other systems reveal a surprising diversity of thermodynamic signatures, emphasizing the delicate balance of contributions to the free energy of formation of RNA tertiary structure.

Entropy Driving the Mg2+-Induced Folding of TPP Riboswitch RNA

“…These observations have implications for how Mg 2+ binding in the aptamer predisposes the folded conformations and favors ligand recognition. We also hypothesize that such free energy changes result from partially folded RNA interaction with Mg 2+ , totally different with temperature-induced unfolding measurements (Figure S5) mainly contributed by enthalpy, i.e., hydrogen bond, base stacking, and other interactions. ,, The results we observed here are in good agreement with entropy change (Δ S ) for the TPP riboswitch ligand binding under multiple Mg 2+ concentrations , or entropy change (Δ S ) for the temperature-induced RNA unfolding experiments at different Mg 2+ . − , The entropy-driven Mg 2+ -induced folding was also reported for a few simple RNAs. − ,, These previous studies for simpler RNAs, together with our results for a more complex riboswitch RNA, identify that being entropy-driven is a characteristic property of Mg 2+ -induced RNA folding.…”

“…5,10,11 The results we observed here are in good agreement with entropy change (ΔS) for the TPP riboswitch ligand binding under multiple Mg 2+ concentrations 12,43 or entropy change (ΔS) for the temperatureinduced RNA unfolding experiments at different Mg 2+ . [5][6][7]11 The entropy-driven Mg 2+ -induced folding was also reported for a few simple RNAs. [5][6][7]11,12 These previous studies for simpler RNAs, together with our results for a more complex riboswitch RNA, identify that being entropy-driven is a characteristic property of Mg 2+ -induced RNA folding.…”

“…The dynamic molecules were informative ones, which were characterized using a two-state HMM and transition density plot (TDP) to explore the conformational switches between states. , There are two ways to visualize the transition density plot in the field of single-molecule studies. (i) One way generates the TDP from all trajectories (including static and dynamic traces) , and (ii) one visualizes this plot only by the transition events of dynamic trajectory. ,, Here, we use the latter strategy to obtain this plot to show the transition events. HMM analysis and TDP at different Mg 2+ indicate that these molecules transit between high-to-low- and low-to-high-FRET states at a rate of ∼0.5 s –1 (Figures a and a–e).…”

“…As Δ G = Δ H – T Δ S , the reduction of Δ G can be achieved either by reducing enthalpy (Δ H ) or by increasing entropy (Δ S ). As widely believed, Mg 2+ can alleviate the enhanced Coulombic interaction associated with a more compact RNA, ,, and it plays an enthalpy-dominated role. On the contrary, some studies support that entropy change is the primary source responsible for stabilizing RNA with Mg 2+ . − ,, However, few reports on the direct thermodynamic characterization of complex RNA folding in the presence of Mg 2+ , and the detailed mechanism of the Mg 2+ -induced RNA folding has not been clarified.…”

“…38,39 There are two ways to visualize the transition density plot in the field of single-molecule studies. (i) One way generates the TDP from all trajectories (including static and dynamic traces) 5,6 and (ii) one visualizes this plot only by the transition events of dynamic trajectory. 1,7,8 Here, we use the latter strategy to obtain this plot to show the transition events.…”