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Nucleic Acids Research, 2003, Vol. 31, No. 10 2595-2600
© 2003 Oxford University Press

Zinc-dependent cleavage in the catalytic core of the hammerhead ribozyme: evidence for a pH-dependent conformational change

Emily J. Borda, John C. Markley and Snorri Th. Sigurdsson

Department of Chemistry, University of Washington, Seattle, WA 98195-1700, USA

*To whom correspondence should be addressed. Tel: +1 206 616 8276; Fax: +1 206 685 8665; Email: sigurdsson{at}chem.washington.edu

We have characterized a novel Zn2+-catalyzed cleavage site between nucleotides C3 and U4 in the catalytic core of the hammerhead ribozyme. In contrast to previously described divalent metal-ion-dependent cleavage of RNA, U4 cleavage is only observed in the presence of Zn2+. This new cleavage site has an unusual pH dependence, in that U4 cleavage products are only observed above pH 7.9 and reach a maximum yield at about pH 8.5. These data, together with the fact that no metal ion-binding site is observed in proximity to the U4 cleavage site in either of the crystal structures, point toward a pH-dependent conformational change in the hammerhead ribozyme. We have described previously Zn2+-dependent cleavage between G8 and A9 in the hammerhead ribozyme and have discovered that U4 cleavage occurs only after A9 cleavage. To our knowledge, this is the first example of sequential cleavage events as a possible regulatory mechanism in ribozymes.


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