NMR and MD studies of the temperature-dependent dynamics of RNA YNMG-tetraloops

Abstract

In a combined NMR/MD study, the temperature-dependent changes in the conformation of two members of the RNA YNMG-tetraloop motif (cUUCGg and uCACGg) have been investigated at temperatures of 298, 317 and 325 K. The two members have considerable different thermal stability and biological functions. In order to address these differences, the combined NMR/MD study was performed. The large temperature range represents a challenge for both, NMR relaxation analysis (consistent choice of effective bond length and CSA parameter) and all-atom MD simulation with explicit solvent (necessity to rescale the temperature). A convincing agreement of experiment and theory is found. Employing a principle component analysis of the MD trajectories, the conformational distribution of both hairpins at various temperatures is investigated. The ground state conformation and dynamics of the two tetraloops are indeed found to be very similar. Furthermore, both systems are initially destabilized by a loss of the stacking interactions between the first and the third nucleobase in the loop region. While the global fold is still preserved, this initiation of unfolding is already observed at 317 K for the uCACGg hairpin but at a significantly higher temperature for the cUUCGg hairpin.

Figure 1.

Order Parameters S² of the 14 nt cUUCGg hairpin (left of the diagrams) extracted from NMR ¹³C relaxation data (open diamonds) and from a MD trajectory (squares) for the nucleobase vectors C6H6/C8H8 (left) and the ribose vectors C1′H1′ (right) at temperatures of 298 K (top), 317 K (middle) and 360 K (bottom). The loop region is shaded in gray. For the highest temperature of 360 K, NMR relaxation data are not available due to stability reasons.

Figure 2.

Order Parameters S² of the 30-nt (NMR) and 14-nt (MD) uCACGg hairpin (left of the diagrams) extracted from NMR ¹³C relaxation data (open diamonds) and from a MD trajectory (squares) for the nucleobase vectors C6H6/C8H8 (left) and the ribose vectors C1′H1′ (right) at temperatures of 298 K (top), of 317 K (middle) and at the onset of the hairpin melting at 325 K in NMR and 360 K in MD, respectively. The loop region is shaded in gray.

Figure 3.

The cUUCGg (left) and uCACGg (right) hairpin configurations at 298 K (top), 317 K (middle) and at 360 K (bottom) projected on the first two PCA eigenvectors. The PCA analysis was performed on the atoms coordinates of residues C5–G10/U13–G18. Shown are the projections every 20 ps for five different time intervals: 0–10 ns (black); 10–20 (red); 20–30 ns (green); 30–40 ns (blue) and 40–50 (beige).

Figure 4.

Representative conformations of the PCA clusters (Figure 3) from the MD simulations at 298 K, 317 K and 360 K. Dashed lines around residue labels indicate that the residues point into the solvent. Parallel lines between residues indicate base pair interactions and thick lines stacking interactions between nucleobases.

Figure 5.

Dynamic behavior of the loop-out residues U7 (top) and A15 (bottom) at 298 K extracted from the molecular dynamics simulation trajectory revealed by the torsional angle χ.