Properties of a Hammerhead Ribozyme with Deletion of Stem II

抄録

The properties of a mutant hammerhead ribozyme system, which consists of two RNA oligomer strands and in which stem II is deleted (replaced with a UUUU loop), are described. The effects of temperature, pH, and metal ions on the cleavage reaction were similar to those for the parent ribozyme with stem II. The mutant ribozyme showed a much lower cleavage rate (k_cat=0.04min^-1) in the presence of 10mM MgCl₂, where the parent ribozyme showed full cleavage activity. However, increasing the concentration of MgCl₂ from 10 to 100mM restored the cleavage activity of the mutant ribozyme to the original level (k_cat=0.2min^-1). CD titration experiments with MgCl₂ using a noncleavable substrate were carried out. Deletion of stem II resulted in an about 20-fold reduction of the apparent Mg²⁺ binding affinity when the Mg²⁺ concentrations of half-saturation are compared. The results were analyzed by curve-fitting analysis and compared with those for the parent ribozyme. The analysis showed that the Mg²⁺ concentration dependence data in CD and cleavage experiments for the mutant enzyme can be explained by a two Mg²⁺ ion binding mechanism. These results suggest that stem II is important for maintaining the conformation of the catalytic core suitable for Mg²⁺ binding.