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Comparison of the hammerhead cleavage reactions stimulated by monovalent and divalent cations

Published online by Cambridge University Press:  09 April 2001

JESSICA L. O'REAR
Affiliation:
Department of Biochemistry, Stanford University, Stanford, California 94305-5307, USA
SHENGLONG WANG
Affiliation:
Department of Biochemistry, Stanford University, Stanford, California 94305-5307, USA NuGen Technologies Co., 2462 Wyabditte St., Mountain View, California 94043, USA
ANDREW L. FEIG
Affiliation:
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA Department of Chemistry, Indiana University, 800 E. Kirkwood Avenue, Bloomington, Indiana 47405, USA
LEONID BEIGELMAN
Affiliation:
Ribozyme Pharmaceuticals Inc., Boulder, Colorado 80301, USA
OLKE C. UHLENBECK
Affiliation:
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA
DANIEL HERSCHLAG
Affiliation:
Department of Biochemistry, Stanford University, Stanford, California 94305-5307, USA
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Abstract

Although the hammerhead reaction proceeds most efficiently in divalent cations, cleavage in 4 M LiCl is only ∼10-fold slower than under standard conditions of 10 mM MgCl2 (Murray et al., Chem Biol, 1998, 5:587–595; Curtis & Bartel, RNA, 2001, this issue, pp. 546–552). To determine if the catalytic mechanism with high concentrations of monovalent cations is similar to that with divalent cations, we compared the activities of a series of modified hammerhead ribozymes in the two ionic conditions. Nearly all of the modifications have similar deleterious effects under both reaction conditions, suggesting that the hammerhead adopts the same general catalytic structure with both monovalent and divalent cations. However, modification of three ligands previously implicated in the binding of a functional divalent metal ion have substantially smaller effects on the cleavage rate in Li+ than in Mg2+. This result suggests that an interaction analogous to the interaction made by this divalent metal ion is absent in the monovalent reaction. Although the contribution of this divalent metal ion to the overall reaction rate is relatively modest, its presence is needed to achieve the full catalytic rate. The role of this ion appears to be in facilitating formation of the active structure, and any direct chemical role of metal ions in hammerhead catalysis is small.

Type
Research Article
Copyright
© 2001 RNA Society

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