Structures and Energetics of Four Adjacent G·U Pairs That Stabilize an RNA Helix

Consecutive G·U base pairs inside RNA helices can be destabilizing, while those at the ends of helices are thermodynamically stabilizing. To determine if this paradox could be explained by differences in base stacking, we determined the high-resolution (1.32 Å) crystal structure of (5′-GGUG­GCUG­UU-3′)₂ and studied three sequences with four consecutive terminal G·U pairs by NMR spectroscopy. In the crystal structure of (5′-GGUG­GCUG­UU-3′)₂, the helix is overwound but retains the overall features of <i>A</i>-form RNA. The penultimate base steps at each end of the helix have high base overlap and contribute to the unexpectedly favorable energetic contribution for the 5′-GU-3′/3′-UG-5′ motif in this helix position. The balance of base stacking and helical twist contributes to the positional dependence of G·U pair stabilities. The energetic stabilities and similarity to <i>A</i>-form RNA helices suggest that consecutive G·U pairs would be recognized by RNA helix binding proteins, such as Dicer and Ago. Thus, these results will aid future searches for target sites of small RNAs in gene regulation