The different role of high-affinity and low-affinity metal ions in cleavage by a tertiary stabilized Cis hammerhead ribozyme from tobacco ringspot virus

The aim of this study was to investigate the dependence of the observed cleavage rates (k_obs) of a tertiary stabilized hammerhead ribozyme (tsHHRz) and of a minimal hammerhead ribozyme (mHHRz), both derived from tobacco ringspot virus, on the type and concentration of divalent metal ions in order to interpret the functional role of high-affinity ions detected by electron paramagnetic resonance (EPR). To measure the fast cleavage of the cis tsHHRz, a new method using chemically synthesized fluorescent-labeled RNAs has been developed. The tsHHRz cleavage rate is up to 20-fold faster than that of the mHHRz under similar conditions. The presence of Mn²⁺ ions leads to a 60-fold faster cleavage than in the presence of Mg²⁺ ions. The functional role of the high-affinity ion was evaluated using neomycin B inhibition studies. Neomycin B reduces the cleavage activity of both ribozymes but the inhibitory effect on tsHHRz is much weaker than that on the mHHRz. EPR data had shown that neomycin B displaces both low-affinity and high-affinity Mn²⁺ ions from the mHHRz, but only low-affinity ions from tsHHRz. Inhibition of the tsHHRz activity may be due to the displacement of weakly bound Me²⁺ ions required for the local folding leading to cleavage, whereas both the high-affinity ion required for folding and the weakly bound ions are replaced in the mHHRz. The high-affinity metal ion is required for the stabilization of the global HHRz structure, but is not involved in catalysis or stabilization of the transient state.