Published online 9 August 2004
Nucleic Acids Research, Vol. 32 No. 14 © Oxford University Press 2004; all rights reserved
Importance in catalysis of a magnesium ion with very low affinity for a hammerhead ribozyme
1 Gene Function Research Center and 2 iGENE Therapeutics, Inc., National Institute of Advanced Industrial Science and Technology (AIST), Central 4, 1-1-1 Higashi, Tsukuba Science City 305-8562, Japan and 3 Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Hongo, Tokyo 113-8656, Japan
* To whom correspondence should be addressed at Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Hongo, Tokyo 113-8656, Japan. Tel: +81 3 5841 8828 or +81 29 861 3015; Fax: +81 29 861 3019; Email: taira{at}chembio.t.u-tokyo.ac.jp
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
Received December 16, 2003; Revised and Accepted July 21, 2004
Available evidence suggests that Mg2+ ions are involved in reactions catalyzed by hammerhead ribozymes. However, the activity in the presence of exclusively monovalent ions led us to question whether divalent metal ions really function as catalysts when they are present. We investigated ribozyme activity in the presence of high levels of Mg2+ ions and the effects of Li+ ions in promoting ribozyme activity. We found that catalytic activity increased linearly with increasing concentrations of Mg2+ ions and did not reach a plateau value even at 1 M Mg2+ ions. Furthermore, this dependence on Mg2+ ions was observed in the presence of a high concentration of Li+ ions. These results indicate that the Mg2+ ion is a very effective cofactor but that the affinity of the ribozyme for a specific Mg2+ ion is very low. Moreover, cleavage by the ribozyme in the presence of both Li+ and Mg2+ ions was more effective than expected, suggesting the existence of a new reaction pathway—a cooperative pathway—in the presence of these multiple ions, and the possibility that a Mg2+ ion with weak affinity for the ribozyme is likely to function in structural support and/or act as a catalyst.
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