RIBOZYME-MEDIATED CLEAVAGE OF A SUBSTRATE-ANALOG CONTAINING AN INTERNUCLEOTIDE-BRIDGING 5'-PHOSPHOROTHIOATE - EVIDENCE FOR THE SINGLE-METALMODEL

An oligonucleotide substrate containing a 5'-bridging phosphorothioate linkage adjacent to a ribonucleotide has been used to investigate the cleavage mechanism of the hammerhead ribozyme and to probe the cataly tic roe of the metal cofactor(s). Specifically, we tested the hypothes is that a second metal interacts with the 5'-leaving group to facilita te the cleavage event. To this end, we have examined the ribozyme-medi ated cleavage activity of the phosphorothioate substrate at pH 7.5 wit h a series of divalent metals in both the presence and absence of the polycation spermine, The cleavage products are found to be the same as for the native sequence under a variety of reaction conditions, The i nfluence of divalent metal ion concentration, temperature, and pH on t he cleavage rate has also been examined for both the oxo linkage and t he thio analogue. Spermine (but not spermidine or NaCl) is shown to su pport efficient cleavage of the thio analogue in the absence [5 mM eth ylenediaminetetraacetic acid (EDTA)] of a divalent metal cofactor. The cleavage of the oxo linkage exhibits a solvent deuterium isotope effe ct of 3.6, but a similar effect is not observed with the thio analogue . The pseudo-first-order rate constants for cleavage off the thio anal ogue in the presence of 10 mM Mg2+ or Mn2+ at pH 7.5 are 65 and 82 x 1 0(-3) min(-1), respectively. The native oxo linkage is cleaved at esse ntially the same rate as the thio analogue (35 and 97 x 10(-3) min(-1) for Mg2+ and Mn2+, respectively). The absence of an appreciable thio effect and the lack of a preference for either Mg2+ or Mn2+ provides c ompelling evidence that the metal cofactor does not interact with the 5'-thioanion (cry oxyanion) leaving group in the transition state. The se rate comparisons additionally reveal that the departure of the 5'-l eaving group is not the rate-limiting step of the cleavage reaction ca talyzed by the hammerhead ribozyme.