Fast folding of an RNA tetraloop on a rugged energy landscape detected by a stacking-sensitive probe

Abstract

We investigate the microsecond-timescale kinetics of the RNA hairpin ga*cUUCGguc. The fluorescent nucleotide 2-aminopurine (a*) reports mainly on base stacking. Ten kinetic traces and the temperature denaturation curve are globally fitted to four-state models of the free-energy surface. In the best-fitting sequential model, the hairpin unfolds over successively larger barriers in at least three stages: stem fraying and increased base-stacking fluctuations; concerted loss of hydrogen bonding and partial unstacking; and additional unstacking of single strands at the highest temperatures. Parallel and trap models also provide adequate fits: such pathways probably also play a role in the complete free-energy surface of the hairpin. To interpret the model states structurally, 200 ns of molecular dynamics, including six temperature-jump simulations, were run. Although the sampling is by no means comprehensive, five different states were identified using hydrogen bonding and base stacking as reaction coordinates. The four to five states required to explain the experiments or simulations set a lower limit on the complexity of this small RNA hairpin's energy landscape.

Figure 1

Experimental thermal denaturation and temperature-jump data fitted by a four-state sequential model. (A) Thermodynamic denaturation data and model fit (red/light gray). (B–D) Relative fluorescence lifetime change and model fit at three of the five temperatures measured (see also Supporting Material). (E–G) Relative fluorescence intensity change and model fit. A parallel model, which does not fit quite as well, is shown in blue for comparison (for details on this and a trap model, see Supporting Material).

Figure 2

State populations and free energies: N, native state; E, frayed state; U, unfolded state without native hydrogen bonding but with residual stacking; S, single-stranded unstacked denatured state. (A) Populations from the four-state sequential model. (B) Free energies and structures of the four fitted states at 353 K on a two-dimensional free-energy landscape, projected onto the sequential and parallel models. Although the sequential model (solid arrows) provides the best one-dimensional fit, parallel processes (dotted arrows) and traps (see Supporting Material) can also make contributions to the complex dynamics of this RNA hairpin.

Figure 3

Molecular dynamics simulation structural results are shown. (A) Representative conformations of the native, frayed, unfolded, and fully unstacked states. Arrows in Fig. 4 indicate the times at which these configurations were sampled. Blue, loop; red, stacked stem bases; ochre, unstacked stem bases; blue dotted lines, native hydrogen bonds. (B) Conformational probability plot of stem hydrogen bonds and stem stacking interactions color-coded by temperature: black, 273 K equilibrium trajectory; red, after jumps to 300 K; green, after jumps to 333 K; blue, after jumps to 498 K. The outermost contour for each ensemble is at 50% of the highest-probability contour. At least five conformational ensembles or subensembles are detected by our limited MD sampling.

Figure 4

Molecular dynamics trajectory results are shown. Equilibrium molecular dynamics, at 273 K, is shown at the upper left; loop hydrogen bonds (green/bottom trace) and loop stacking (not shown) made relatively small contributions and were not analyzed further. The remaining three panels show simulations of three temperature jumps starting at time t = 0. Native stem base stacking (red/light) and native stem hydrogen bonding (blue/dark) are shown, with each time point averaged over a 0.5-ns window. The arrows indicate the point at which the conformations shown in Figs. 2 and 3A were sampled. The T-jumps are each an average of two trajectories with different random initial conditions. The individual trajectories are shown in the Supporting Material.