Identification and Characterization of a Divalent Metal Ion-Dependent Cleavage Site in the Hammerhead Ribozyme

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• 作者：John C. Markley ; Fré ; dé ; ric Godde ; and Snorri Th. Sigurdsson
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：November 20, 2001
• 年：2001
• 卷：40
• 期：46
• 页码：13849 - 13856
• 全文大小：139K
• 年卷期：v.40,no.46(November 20, 2001)
• ISSN：1520-4995

文摘

We describe a new RNA cleavage motif, found in the hammerhead ribozyme. Cleavage occursbetween nucleotides G8 and A9, yielding a free 5'-hydroxyl group and a 2',3'-cyclic phosphate. Thiscleavage is dependent upon divalent metal ions and is the first evidence for a metalloribozyme known toshow preference for Zn²⁺. Cleavage is also observed in the presence of Ni²⁺, Co²⁺, Mn²⁺, Cd²⁺, andPb²⁺, while negligible cleavage was detected in the presence of the alkaline-earth metal ions Mg²⁺, Ca²⁺,Sr²⁺, and Ba²⁺. A linear relationship between the logarithm of the rate and pH was observed for theZn²⁺-dependent cleavage, which is indicative of proton loss in the cleavage mechanism, either prior to orin the rate-determining step. We postulate that a zinc hydroxide complex, bound to the known A9/G10.1metal ion binding site, abstracts the proton from the 2'-hydroxyl group of G8, which attacks the A9phosphate and initiates cleavage. This hypothesis is supported by a previously reported crystal structure[Murray, J. B., Terwey, D. P., Maloney, L., Karpeisky, A., Usman, N., Beigelman, L., and Scott, W. G.(1998) Cell 92, 665-673], which shows the conformation required for RNA cleavage and proximity ofthe 2'-hydroxyl group to the metal ion complex.