Substitution of the 2′-hydroxyl group at position 2.1 by an amino group interferes with Mg2+ binding and efficient cleavage by hammerhead ribozyme

被引:5
作者
Sioud, M [1 ]
Leirdal, M [1 ]
机构
[1] Norwegian Radium Hosp, Dept Immunol, Inst Canc Res, N-0310 Oslo, Norway
关键词
D O I
10.1006/bbrc.1999.1236
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Recently we have demonstrated that hammerhead ribozymes can be fully substituted with 2'-amino pyrimidines without detriment to the catalytic activity, provided that positions 2.2 and/or 2.1 are not modified. We now report on the potential molecular mechanisms by which 2'-amino groups at these positions inhibit the ribozyme cleavage activity. In the presence of Mg2+, the 2'-amino modification at positions 2.2 and/or 2.1 had no significant effect on substrate binding. Detailed analysis of the ribozyme initial cleavage rates in the presence of various Mg2+ concentrations indicated that Mg2+ binding is inhibited by the 2'-amino group at position 2.1. Furthermore, preannealed substrate molecules to the modified ribozyme are not effectively cleaved upon Mg2+ addition, indicating an alteration of the ribozyme cleavage step. Surprisingly, the cleavage activity of the modified ribozymes was substantially increased when Mg2+ ions were replaced by the thiophilic Mn2+ ions, whereas only a moderate cleavage enhancement occurred with its unmodified version. Taken together, our findings indicate that changes in the sugar at position 2.1 alter Mg2+-promoting ribozyme cleavage. (C) 1999 Academic Press.
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页码:461 / 466
页数:6
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