MAGNESIUM-MEDIATED CONVERSION OF AN INACTIVE FORM OF A HAMMERHEAD RIBOZYME TO AN ACTIVE COMPLEX WITH ITS SUBSTRATE - AN INVESTIGATION BY NMR-SPECTROSCOPY

The effects of magnesium ions on a 32-mer ribozyme (R32) were examined by high resolution NMR spectroscopy, In solution, R32 (without its su bstrate) consisted of a GAAA loop, stem II, a non-Watson-Crick 3-base pair duplex and a 4-base pair duplex that included a wobble G:U base p air, When an uncleavable substrate RNA (RdC11) was added to R32 withou t Mg2+ ions, a complex did not form between R32 and RdC11 because the substrate recognition regions of R32 formed intramolecular base pairs (the recognition arms were closed), By contrast, in the presence of Mg 2+ ions, the R32-RdC11 complex was formed. Moreover, titration of mixt ures of R32 and RdC11 with Mg2+ ions also induced the ribozyme-substra te interaction, Elevated concentrations (1.0 hi) of monovalent Na+ ion s could not induce the formation of the R32-RdC11 complex. These data suggest that Mg2+ ions are not only important as the true catalysts in the function of ribozyme-type metalloenzymes, but they also induce th e structural change in the R32 hammerhead ribozyme that is necessary f or establishment of the active form of the ribozyme-substrate complex.