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.