Structure of an RNA Hairpin Loop with a 5‘-CGUUUCG-3‘ Loop Motif by Heteronuclear NMR Spectroscopy and Distance Geometry

Abstract

Structural features of a 19-nucleotide RNA hairpin loop (5'-GGCGUACGUUUCGUACGCC-3'), a loop motif which occurs in eukaryotic 18S rRNA, have been derived using multidimensional heteronuclear NMR spectroscopy in combination with local conformational analysis and torsion angle distance geometry followed by restrained energy minimization. A method to obtain both the 3JC4'P3' and 3JC4'P5' coupling constants from a set of spin-echo difference constant time HSQC spectra is introduced, and it is shown how these couplings can be assigned to the backbone angles beta and epsilon. A total of 280 distance constraints as well as 132 homo- and heteronuclear three-bond scalar coupling constants were derived from the NMR data. The structure which has been determined is a pentaloop rather than a triloop with no base pairing between G8 and C12. G8 is pointed to the minor groove where it forms a base triplet with C7-G13 that is further stabilized by hydrogen bonding to the 2'-hydroxyl group of C7. C12 is directed to the major groove where its conformation is stabilized by hydrogen bonding between O2 and HO2'. The NMR data suggest two possible, interconverting conformations with stacking of bases U10-G8 or U11-C7. Overall, the loop provides a variety of interaction sites for RNA or protein interactions.

NMR Characterization of an Oligonucleotide Model of the MiR-21 Pre-Element

“…Our NMR-derived model, in conjunction with the imino proton spectra of 2 and related hairpins, suggest a direct hydrogen bonding interaction between these nucleotides across the major groove. The orientation of U18 is similar to a feature seen in the solution structure of a five-nucleotide hairpin loop modeled after a loop in the 18S ribosomal RNA [48]. In that case, a cytosine at the 3′ end of the loop sequence is oriented toward the major groove of the adjacent double helix.…”

“…The predicted secondary structure of the loop, formally a five-nucleotide loop with a bulged nucleotide adjacent to the closing base pair, is unlike hairpin loops that have been studied previously by NMR [48]–[50]. Much of the structural analysis of hairpin loops has been directed to four-nucleotide loops such as the stable UNCG tetraloops [51] and the GNRA loop motif [52].…”

Solution structure of stem-loop α of the hepatitis B virus post-transcriptional regulatory element

“…The NMR spectra were processed with VNMR (Varian Assoc., Palo Alto, USA) and analysed using the program CARA (33). Chemical shifts were referenced as described previously (34). Resonance assignment of the exchangeable and non-exchangeable protons of HSLA and HSLAap was performed as previously described in (31,32,34–36) using ( 1 H, 15 N)-HSQC (37), ( 1 H, 15 N)-CPMG-NOESY (38), ( 1 H, 1 H)-H5(C5C4N)H (C,U) (39), ( 1 H, 1 H)-HCCNH-TOCSY (G) (40), ( 1 H, 1 H)-HCCH-TOCSY (A) (41), ( 1 H, 1 H)-HCCH-COSY (C,U) (42), ( 1 H, 15 N)-2bond-HSQC (43), ( 1 H, 13 C)-H(N)CO (44), ( 1 H, 13 C)-HSQC (37,45), 3D ( 1 H, 1 H, 13 C)-HCCH-COSY (sugar), 3D ( 1 H, 1 H, 13 C)-HCCH-TOCSY (sugar) (42) and ( 1 H, 15 N)-HCN (46).…”

A novel cGUUAg tetraloop structure with a conserved yYNMGg-type backbone conformation from cloverleaf 1 of bovine enterovirus 1 RNA

“…The NMR spectra were processed with VNMR (Varian Assoc., Palo Alto) and analysed using the program XEASY (20). Chemical shifts were referenced as described previously (21). Resonance assignment of the exchangeable and non-exchangeable protons of BEVSLDap was performed using ( 1 H, 15 N)-HSQC (22), ( 1 H, 15 N)-CPMG-NOESY (23), ( 1 H, 1 H)-H5(C5C4N)H (C,U) (24), ( 1 H, 1 H)-HCCNH-TOCSY (G) (25), ( 1 H, 1 H)-HCCH-TOCSY (A) (26), ( 1 H, 1 H)-HCCH-COSY (C,U) (27), ( 1 H, 15 N)-2bond-HSQC (28), ( 1 H, 13 C)-H(N)CO (29), ( 1 H, 13 C)-HSQC, 3D ( 1 H, 1 H, 13 C)-HCCH-COSY (sugar), 3D ( 1 H, 1 H, 13 C)-HCCH-TOCSY (sugar) (27) and ( 1 H, 15 N)-HCN (30).…”