Mg(2+)-dependent conformational changes in the hammerhead ribozyme

Biochemistry
M MengerD Pörschke

Abstract

Conformational changes of the hammerhead ribozyme were examined by fluorescence changes of 2-aminopurine riboside incorporated either in the substrate or in the ribozyme. Fluorescence changes could be observed for both the substituted substrate and ribozyme upon complex formation, indicating a different environment for the 2-aminopurine in the complex. Ribozyme-substrate constructs for ciscleavage containing 2-aminopurine at various sites were used for the determination of binding constants of Mg2+ and Ca2+. Depending upon the site of 2-aminopurine substitutions, the fluorescence intensity upon addition of Mg2+ or Ca2+ was reduced by 0-50%. The measurements were performed in high ionic strength buffers such that base pairing in the helical regions is expected to be complete. With three of the ribozymes, the dependence of the fluorescence emission as a function of Mg2+ concentration could be fitted by single binding processes, whereas for the two remaining ribozymes a second binding process needed to be included. The binding constants range from 7600 M-1 down to 12 M-1 in 75 mM Tris-HCl (pH 7.5) and indicate the presence of multiple binding sites in the ribozymes with varying degrees of affinity toward the metal ions. Mg2+ bindi...Continue Reading

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