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Ribozyme cleavage of a 2′,5′-phosphodiester linkage: Mechanism and a restricted divalent metal-ion requirement

  • I-HUNG SHIH (a1) and MICHAEL D. BEEN (a1)

Abstract

The natural substrate cleaved by the hepatitis delta virus (HDV) ribozyme contains a 3′,5′-phosphodiester linkage at the cleavage site; however, a 2′,5′-linked ribose-phosphate backbone can also be cleaved by both trans-acting and self-cleaving forms of the HDV ribozyme. With substrates containing either linkage, the HDV ribozyme generated 2′,3′-cyclic phosphate and 5′-hydroxyl groups suggesting that the mechanisms of cleavage in both cases were by a nucleophilic attack on the phosphorus center by the adjacent hydroxyl group. Divalent metal ion was required for cleavage of either linkage. However, although the 3′,5′-linkage was cleaved slightly faster in Ca2+ than in Mg2+, the 2′,5′-linkage was cleaved in Mg2+ (or Mn2+) but not Ca2+. This dramatic difference in metal-ion specificity is strongly suggestive of a crucial metal-ion interaction at the active site. In contrast to the HDV ribozymes, cleavage at a 2′,5′-phosphodiester bond was not efficiently catalyzed by the hammerhead ribozyme. The relaxed linkage specificity of the HDV ribozymes may be due in part to lack of a rigid binding site for sequences 5′ to the cleavage site.

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Corresponding author

Reprint requests to: Michael D. Been, Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27705, USA; e-mail: been@biochem.duke.edu.

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Ribozyme cleavage of a 2′,5′-phosphodiester linkage: Mechanism and a restricted divalent metal-ion requirement

  • I-HUNG SHIH (a1) and MICHAEL D. BEEN (a1)

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