RNA Internal Loops with Tandem AG Pairs: The Structure of the 5′GAGU/3′UGAG Loop Can Be Dramatically Different from Others, Including 5′AAGU/3′UGAA

Abstract

Thermodynamic stabilities of 2 × 2 nucleotide tandem AG internal loops in RNA range from −1.3 to +3.4 kcal/mol at 37 °C and are not predicted well with a hydrogen-bonding model. To provide structural information to facilitate development of more sophisticated models for the sequence dependence of stability, we report the NMR solution structures of five RNA duplexes: (rGACGAGCGUCA)2, (rGACUAGAGUCA)2, (rGACAAGUGUCA)2, (rGGUAGGCCA)2, and (rGACGAGUGUCA)2. The structures of these duplexes are compared to that of the previously solved (rGGCAGGCC)2 (Wu, M., SantaLucia, J., Jr., and Turner, D. H. (1997) Biochemistry 36, 4449−4460). For loops bounded by Watson−Crick pairs, the AG and Watson−Crick pairs are all head-to-head imino-paired (cis Watson−Crick/Watson−Crick). The structures suggest that the sequence-dependent stability may reflect non-hydrogen-bonding interactions. Of the two loops bounded by G-U pairs, only the 5′UAGG/3′GGAU loop adopts canonical UG wobble pairing (cis Watson−Crick/Watson−Crick), with AG pairs that are only weakly imino-paired. Strikingly, the 5′GAGU/3′UGAG loop has two distinct duplex conformations, the major of which has both guanosine residues (G4 and G6 in (rGACGAGUGUCA)2) in a syn glycosidic bond conformation and forming a sheared GG pair (G4-G6*, GG trans Watson−Crick/Hoogsteen), both uracils (U7 and U7*) flipped out of the helix, and an AA pair (A5-A5*) in a dynamic or stacked conformation. These structures provide benchmarks for computational investigations into interactions responsible for the unexpected differences in loop free energies and structure.