The Different Role of High-Affinity and Low-Affinity Metal Ions in Cleavage by a Tertiary Stabilized CisHammerhead Ribozyme from Tobacco Ringspot Virus.

The aim of this study was to investigate the dependence of the observed cleavage rates (kobs) 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 cistsHHRz, 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 Mn2ions leads to a 60-fold faster cleavage than in the presence of Mg2ions. 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 Mn2ions from the mHHRz, but only low-affinity ions from tsHHRz. Inhibition of the tsHHRz activity may be due to the displacement of weakly bound Me2ions 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. [ABSTRACT FROM AUTHOR]