Mg2+-Dependent Conformational Changes in the Hammerhead Ribozyme

• Published in: Biochemistry (Easton) 1996-11, Vol.35 (47), p.14710-14716
• Main Authors: Menger, Marcus, Tuschl, Thomas, Eckstein, Fritz, Porschke, Dietmar
• Format: Article
• Language: English
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi960440w

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 cis-cleavage 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+ binding constants determined in the same buffer from the Mg2+ dependence of the cleavage rate are of the order of 100 M-1; thus, Mg2+ sites directly involved in catalysis are of intermediate affinity. The ribozyme containing 2-aminopurine in loop III demonstrated the highest binding constant whereas the ribozyme with a 2-aminopurine next to a 2‘-deoxy-2‘-aminocytosine at the cleavage site exhibited only low metal ion affinity. The data obtained for Ca2+ are very similar to those found for Mg2+. This approach provides a first set of data describing a Mg2+ binding topography to hammerhead RNA molecules and should be useful for the analysis of other RNA molecules.