NMR elucidation of the role of Mg2+ in the structure and stability of the conserved RNA motifs of the EMCV IRES element

Abstract

The three dimensional solution structures of a highly conserved 16mer RNA, endowed with a classic ‘GNRA’ tetraloop motif, and a 17mer RNA containing a cytosine-rich heptaloop which is predicted to be a potential receptor for the former RNA, of the I-domain of Encephalomyocarditis virus IRES have been determined by NMR spectroscopy. As Mg²⁺ plays an important role in the activity of the IRES, the corresponding NMR structures of the Mg²⁺ bound RNA complexes have also been determined. These RNA NMR structures, 16mer (21 constraints per residue), 16mer RNA/Mg²⁺ (21 constraints per residue), 17mer (17 constraints per residue) and 17mer RNA/Mg²⁺ (16 constraints per residue), were calculated to a high degree of precision with low RMSDs and low clash scores represent, to the best our knowledge, the first structures of a type II picornavirus IRES. Conformational analysis of the average structure showed that the RNAs and their Mg²⁺ complexes adopt characteristic A-form helical structures, stabilised by canonical and non-canonical interactions in both the stem and loop regions. The GCGA tetraloop of the 16mer folds into a standard GNRA conformation, with the structural role of A550 being in the form of a G547.A550 sheared base-pair made up of two hydrogen bonds. Further, the previously uncharacterised AACCCCA heptaloop present in the 17mer forms a compact tertiary loop motif, held together by strong π–π interactions. Analysis of the NMR structures demonstrates that the role of Mg²⁺ is principally to confer enhanced stability to the RNAs whereby the tetra- and heptaloops can achieve optimum conformation for any RNA–RNA interactions which are crucial for understanding the structure–function relationship of the IRES.