Structure refinement for a 24-nucleotide RNA hairpin

Refinement of the NMR-based structure of an RNA hairpin that binds the coat protein of bacteriophage R17 is presented. Properties of the upper stem and loop suggest rapid conformational exchange: (i) the G10,1 and G16,1 imino signals are broad, (ii) residues G7-G16 have a substantial fraction of S-puckered ribose rings and (iii) a primary tetraloop conformation satisfies the distance restraints, but other forms occurred in our molecular dynamics simulations that might be in fast exchange with the primary conformation. Unusual methods were used to assign all of the non-exchangeable protons in the molecule. The results show that structure determinations of 20-30-mer RNAs are practical without isotope labeling. The analysis depended on time-domain deconvolution of 2D NOE spectra, the resolving power of 3D 2-quantum correlation/NOE spectra and dispersively phased ¹H-³¹P-COSY spectra. This work corrects some assignment errors in G1-A4, and reports a full-relaxation matrix refinement (averaging >20 distance restraints per residue). The resolution of the new structure confirms that the unpaired A8 residue is stacked in the stem. It also shows the presence of a two-residue pyrimidine turn and a possible A11· A14 non-canonical pair in the tetra-loop. Finally, it determines an unusual structure for the molecule's strand ends. The latter involves stacking of G3/G2/A23/U24, a probable G3·U22 pair and G1 lies in the major groove. The strand-end structure raises interesting possibilities for unique structures at the junctions of double helical RNA with unpaired regions.