NMR studies of the structure and Mg2+ binding properties of a conserved RNA motif of EMCV picornavirus IRES element

The structure and Mg2+ binding properties of a conserved 75mer RNA motif of the internal ribosome entry site (IRES) element of encephalomyocarditis virus picornavirus have been investigated by 1H-NMR and UV melting experiments. The assignment of the imino proton resonances with characteristic chemical shift dispersion for canonical and non-canonical base pairs confirmed the predicted secondary structure of the 75mer and its fragments. Addition of Mg2+ resulted in a dramatic increase in apparent melting temperature, with the 75mer RNA registering the biggest increase, from 63 to 80°C, thus providing evidence for enhanced stability arising from Mg2+ binding. Similarly, addition of Mg2+ induced selective changes to the chemical shifts of the imino protons of a GCGA tetraloop in the 75mer, that is essential for IRES activity, thereby highlighting a possible structural role for Mg2+ in the folding of the 75mer. Significantly, the same protons show retarded exchange to water solvent, even at elevated temperature, which suggest that Mg2+ induces a conformational rearrangement of the 75mer. Thus, we propose that Mg2+ serves two important roles: (i) enhancing thermodynamic stability of the 75mer RNA (and its submotifs) via nonspecific interactions with the phosphate backbone and (ii) promoting the folding of the 75mer RNA by binding to thel GCGA tetraloop. © Oxford University Press 2004; all rights reserved.