Effects of magnesium ions on two EMCV IRES key activity fragments
Using NMR magnetization transfer from water and ammonia-catalyzed exchange of the imino protons, changes have been monitored in base-pair kinetics induced by Mg2 + in two key activity fragments r(CACCUGGCGACAGGUG) and r(GGCCAAAAGCC) of the encephalomyocarditis virus internal ribosome entry site. For r(CACCUGGCGACAGGUG), the addition of Mg2 + reveals two types of base-pairs: r(U545·A) and r(G546·C), in the first category, have lifetimes only slightly higher in the presence of Mg2 + , whereas their dissociation constants are substantially reduced. This behavior has been termed proximal. The base-pairs r(G553·C) and r(G554·C), in the second category, have lifetimes substantially higher in the presence of Mg2 + , whereas their dissociation constants remain almost constant. This behavior has been termed distal. Mg2 + has a specific effect on r(CACCUGGCGACAGGUG), the magnitude of which is progressively modulated from the proximal region of the 16-mer towards its distal region. For r(GGCCAAAAGCC), an intermediate behavior is found for base-pairs r(G565·C) and r(G572·C). Their lifetimes are slightly higher in the presence of Mg2 + and their dissociation constants are significantly lower, a behavior resembling that of the 16-mer proximal region. These results indicate that Mg2 + diffusively moves around r(GGCCAAAAGCC).
KeywordsBase-pair kinetics EMCV IRES GNRA tetraloop Imino proton exchange Mg2 + NMR RAAA hairpin motif
Julien A. Dupont is a recipient of a studentship from the Fonds pour la formation à la Recherche dans l’Industrie et l’Agriculture. This work was supported by the Belgian National Foundation for Scientific Research and the Catholic University of Louvain (Fonds Spéciaux de Recherche).