Identification of the hammerhead ribozyme metal ion binding site responsible for rescue of the deleterious effect of a cleavage site phosphorothioate

The hammerhead ribozyme crystal structure identified a specific metal ion b inding site referred to as the P9/G10.1 site. Although this metal ion bindi ng site is similar to 20 Angstrom away from the cleavage site, its disrupti on is highly deleterious for catalysis. Additional published results have s uggested that the pro-R-p oxygen at the cleavage site is coordinated by a m etal ion in the reaction's transition state. Herein, we report a study on C d2+ rescue of the deleterious phosphorothioate substitution at the cleavage site. Under all conditions, the Cd2+ concentration dependence can be accou nted for by binding of a single rescuing metal ion. The affinity of the res cuing Cd2+ is sensitive to perturbations at the P9/G10.1 site but not at th e cleavage site or other sites in the conserved core. These observations le d to a model in which a metal ion bound at the P9/G10.1 site in the ground state acquires an additional interaction with the cleavage site prior to an d in the transition state. A titration experiment ruled out the possibility that a second tight-binding metal ion (K-d(Cd) < 10 mu M) is involved in t he rescue, further supporting the single metal ion model. Additionally, wea kening Cd2+ binding at the P9/G10.1 site did not result in the biphasic bin ding curve predicted from other models involving two metal ions. The large stereospecific thio-effects at the P9/G10.1 and the cleavage site suggest t hat there are interactions with these oxygen atoms in the normal reaction t hat are compromised by replacement of oxygen with sulfur. The simplest inte rpretation of the substantial rescue by Cd2+ is that these atoms interact w ith a common metal ion in the normal reaction. Furthermore, base deletions and functional group modifications have similar energetic effects on the tr ansition state in the Cd2+-rescued phosphorothioate reaction and the wild-t ype reaction, further supporting the model that a metal ion bridges the P9/ G10.1 and the cleavage site in the normal reaction (i.e., with phosphate li nkages rather than phosphorothioate linkages). These results suggest that t he hammerhead undergoes a substantial conformational rearrangement to attai n its catalytic conformation. Such rearrangements appear to be general feat ures of small functional RNAs, presumably reflecting their structural limit ations.