RNA tetraloop folding reveals tension between backbone restraints and molecular interactions

In RNA, A-form helices are commonly terminated by tetraloops or 3' dangling ends. Aside from helices themselves, these helix-breaking motifs appear to be among the most frequent and repetitive structural elements of large folded RNAs. We show here that within a frequent type of tetraloop, cGNRAg (G is guanine, N is any base, R is purine, A is adenine), a tension exists between the backbone torsional energy of the loop and the energy contributed by molecular interactions (stacking and pairing). A model in which favorable bond rotamers are opposed by favorable stacking and pairing interactions is consistent with our observation that release of torsional restraints upon conversion of one or more loop riboses to more flexible trimethylene phosphate(s) contributes favorably to the enthalpy of folding. This effect presumably results from improved stacking and hydrogen-bonding interactions upon release of torsional restraints. The most obvious possibility for improving molecular interactions is a repositioning of A, which is proximal to the unfavorable torsion angles in native cGNRAg tetraloops, and which is unstacked on the 3' side and unpaired (it forms a single hydrogen bond with the opposing G). This tension between favorable bond rotamers and favorable molecular interactions may be representative of a general evolutionary strategy to prevent achievement of deep and irreversible thermodynamic wells in folded RNAs. Finally, we observe a simple stacking substructure with conserved geometry and sequence that forms a scaffold for both tetraloops and 3' dangling ends. It seems that simple substructures can build RNA motifs, which combine to establish the fundamental architecture of RNA.