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 Mg ²⁺, 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).