RNA Internal Loops with Tandem AG Pairs: The Structure of the 5′G(UG)under-barU/3′U(GA)under-barG Loop Can Be Dramatically Different from Others, Including 5′A(AG)under-barU/3′U(GA)under-barA

Thermodynamic stabilities of 2 x 2 nucleotide tandem AG internal loops in RNA range from -1.3 to +3.4 kcal/mol at 37 degrees 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: (rGACG (AG) under bar CGUCA)(2), (rGACU (AG) under bar AGUCA)(2), (rGACA (GA) under bar UGUCA)(2), (rGGU (GA) under bar GCCA)(2), and (rGACG (AG) under bar UGUCA)(2). The structures of these duplexes are compared to that of the previously solved (rGGC (AG) under bar GCC)(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'U (GA) under barG/3'G (GA) under barU loop adopts canonical UG wobble pairing (cis Watson-Crick/Watson-Crick), with AG pairs that are only weakly imino-paired. Strikingly, the 5'G (GA) under barU/3'U (GA) under barG loop has two distinct duplex conformations, the major of which has both guanosine residues (G4 and 06 in (rGACG (AG) under bar UGUCA)(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.