著者
Sakamoto Taiichi Kim Mi Hee Kurihara Yasuyuki SASAKI Nobuyuki NOGUCHI Tomoaki KATAHIRA Masato UESUGI Seiichi
出版者
社団法人 日本生化学会
雑誌
The journal of biochemistry (ISSN:0021924X)
巻号頁・発行日
vol.121, no.2, pp.288-294, 1997-02-01
参考文献数
48
被引用文献数
1

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<sub>cat</sub>=0.04min<sup>-1</sup>) in the presence of 10mM MgCl<sub>2</sub>, where the parent ribozyme showed full cleavage activity. However, increasing the concentration of MgCl<sub>2</sub> from 10 to 100mM restored the cleavage activity of the mutant ribozyme to the original level (k<sub>cat</sub>=0.2min<sup>-1</sup>). CD titration experiments with MgCl<sub>2</sub> using a noncleavable substrate were carried out. Deletion of stem II resulted in an about 20-fold reduction of the apparent Mg<sup>2+</sup> binding affinity when the Mg<sup>2+</sup> 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 Mg<sup>2+</sup> concentration dependence data in CD and cleavage experiments for the mutant enzyme can be explained by a two Mg<sup>2+</sup> ion binding mechanism. These results suggest that stem II is important for maintaining the conformation of the catalytic core suitable for Mg<sup>2+</sup> binding.