Nuclear Magnetic Resonance Reveals That GU Base Pairs Flanking Internal Loops Can Adopt Diverse Structures.

RNA thermodynamics play an important role in determining the two- and three-dimensional structures of RNA. Internal loops of the sequence 5′-GMNU/3′-UNMG are relatively unstable thermodynamically. Here, five duplexes with GU-flanked 2 × 2 nucleotide internal loops were structurally investigated to reveal determinants of their instability. The following internal loops were investigated: 5′-GCAU/3′-UACG, 5′-UUCG/3′-GCUU, 5′-GCUU/3′-UUCG, 5′-GUCU/3′-UCUG, and 5′-GCCU/3′-UCCG. Two-dimensional nuclear magnetic resonance spectra indicate the absence of GU wobble base pairing in 5′-GCUU/3′-UUCG, 5′-GUCU/3′-UCUG, and 5′-GCCU/3′-UCCG. The 5′-GCUU/3′-UUCG loop has an unusual conformation of the GU base pairs, in which U’s O2 carbonyl forms a bifurcated hydrogen bond with G’s amino and imino protons. The internal loop of 5′-GUCU/3′-UCUG displays a shifted configuration in which GC pairs flank a U-U pair and several U’s are in fast exchange between positions inside and outside the helix. In contrast, 5′-GCAU/3′-UACG ...