Ribozyme-Mediated Cleavage of a Substrate Analogue Containing an Internucleotide-Bridging 5‘-Phosphorothioate:  Evidence for the Single-Metal Model

Abstract

An oligonucleotide substrate containing a 5'-bridging phosphorothioate linkage adjacent to a ribonucleotide has been used to investigate the cleavage mechanisms of the hammerhead ribozyme and to probe the catalytic role of the metal cofactor(s). Specifically, we tested the hypothesis that a second metal interacts with the 5'-leaving group to facilitate the cleavage event. To this end, we have examined the ribozyme-mediated cleavage activity of the phosphorothioate substrate at pH 7.5 with a series of divalent metal 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 influence of divalent metal ion concentration, temperature, and pH on the cleavage rate also has been examined for both the oxo linkage and the thio analogue. Spermine (but not spermidine or NaCl) is shown to support efficient cleavage of the thio analogue in the absence [5 mM ethylenediaminetetraacetic acid (EDTA)] of a divalent metal cofactor. The cleavage of the oxo linkage exhibits a solvent deuterium isotope effect of 3.6, but a similar effect is not observed with the thio analogue. The pseudo-first-order rate constants for cleavage of the thio analogue in the presence of 10 mM Mg2+ or Mn2+ at pH 7.5 are 65 and 82 x 10(-3) min-1, respectively. The native oxo linkage is cleaved at essentially 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 compelling evidence that the metal cofactor does not interact with the 5'-thioanion (or oxyanion) leaving group in the transition state. These rate comparisons additionally reveal the the departure of the 5'-leaving group is not the rate-limiting step of the cleavage reaction catalyzed by the hammerhead ribozyme.

Observations on catalysis by hammerhead ribozymes are consistent with a two-divalent-metal-ion mechanism

“…2 A) has also been replaced by sulfur. Cleavage of the 5Ј-sulfur substrate is greatly stimulated even in the absence of the ribozyme and divalent metal ions (12). For the sulfur-containing substrate, the cleavage rate is increased by the addition of Mn 2ϩ and Mg 2ϩ , but no significant difference in cleavage activity is observed for these two metal ions.…”

“…Several phosphate oxygens in the ribozyme-substrate complex have been replaced by sulfur (12,(20)(21)(22). These phosphorothioate replacement studies are used to identify oxygen atoms that interact directly with divalent metal ions (23 6 ].…”

“…For the sulfur-containing substrate, the cleavage rate is increased by the addition of Mn 2ϩ and Mg 2ϩ , but no significant difference in cleavage activity is observed for these two metal ions. These data were taken as evidence that a metal ion is not coordinated at the 5Ј-sulfur and, by analogy, also not coordinated at the naturallyoccurring 5Ј-oxygen (12). The absence of a metal ion at this position was taken as direct evidence against a two-metal-ion model, since the two-metal-ion model includes a metal ion coordinated to the 5Ј-oxygen.…”

“…Unlike thiophosphate replacements made in other positions, the 5Ј-sulfur replacement substrate is activated Ϸ10 6 -fold for cleavage (relative to the 5Ј-oxygen substrate) even in the absence of divalent ions (12). This substrate is prone to cleavage even in the absence of ribozyme (12).…”

“…These include an inverse correlation between the pK a values of different metal ions and their relative cleavage activities (7), as well as the effects of phosphorothioate replacements at the 5Ј-internucleotide bridging oxygen (12). In addition, an observed solvent isotope effect has been interpreted as evidence that a proton transfer step does not occur in the transition state for the hammerhead ribozyme (13).…”