Mg2+ modulation of EMCV IRES key activity fragment equilibria and r(G·C) base-pair kinetics

Abstract

NMR magnetization transfer from water and ammonia-catalyzed exchange of the imino proton have been used to probe enhanced thermostability and conformational rearrangements induced by Mg(2+) in two key activity fragments r(CACCUGGCGACAGGUG) and r(GGCCAAAAGCC) of the encephalomyocarditis virus (EMCV) picornavirus internal ribosome entry site (IRES). We have measured some of their r(G*C) base-pair lifetimes and dissociation constants under different MgCl(2) conditions, and we compare them with those of other short RNA duplexes. The RNA fragment r(CACCUGGCGACAGGUG) adopts two topologies, a palindromic duplex with two conformations and a hairpin, whose equilibrium can be monitored: the duplex form is destabilized by Mg(2+) and temperature, a delicate balance wherein the entropic contribution of the free energy helps populate the hairpin state. For both fragments, the opening rates of the r(G*C) pairs are lower in the presence of Mg(2+) and their dissociation constants are smaller or comparable. Analysis of the results suggests that Mg(2+) has a preferential and specific effect on the r(CACCUGGCGACAGGUG) hairpin in the region close to the r(G*C) closing pair of the GCGA tetraloop, and the ion moves diffusively around r(GGCCAAAAGCC), thereby differentiating the GNRA and RAAA hairpin motifs that are both involved in the biological regulation functions of the EMCV IRES.