Experimental and computational studies of the G[UUCG]C RNA tetraloop

In prokaryotic ribosomal RNAs, most UUCG tetraloops are closed by a C-G base-pair. However, this preference is greatly reduced in eukaryotic rRNA species where many UUCG tetraloops are closed by G-C base-pairs. Here, biophysical properties of the CUUCG]G and GUUCG]C tetraloops are compared, using experimental and computational methods. Thermal denaturation experiments are used to derive thermodynamic parameters for the wild-type GUUCG]C tetraloop and variants containing single deoxy substitutions in the loop. A comparison with analogous experiments on the CUUCG]G motif shows that the two RNA species exhibit similar patterns in response to the substitutions, suggesting that their loop structures are similar. This conclusion is supported by NMR data that suggest that the essential UUCG loop structure is maintained in both tetraloops. However, NMR results show that the GUUCG]C loop structure is disrupted prior to melting of the stem; this behavior is in contrast to the two-state behavior of the CUUCG]G molecule. Stochastic dynamics simulations using the GB/SA continuum solvation model, run as a function of temperature, show rare conformational transitions in several GUUCG]C simulations. These results lead to the conclusion that substitution of a G-C for a C-G closing base-pair increases the intrinsic flexibility of the UUCG loop.