Capturing Hammerhead Ribozyme Structures in Action by Modulating General Base Catalysis

Abstract

We have obtained precatalytic (enzyme–substrate complex) and postcatalytic (enzyme–product complex) crystal structures of an active full-length hammerhead RNA that cleaves in the crystal. Using the natural satellite tobacco ringspot virus hammerhead RNA sequence, the self-cleavage reaction was modulated by substituting the general base of the ribozyme, G12, with A12, a purine variant with a much lower pK_a that does not significantly perturb the ribozyme's atomic structure. The active, but slowly cleaving, ribozyme thus permitted isolation of enzyme–substrate and enzyme–product complexes without modifying the nucleophile or leaving group of the cleavage reaction, nor any other aspect of the substrate. The predissociation enzyme-product complex structure reveals RNA and metal ion interactions potentially relevant to transition-state stabilization that are absent in precatalytic structures. Enzymes use variations of a few standard approaches to catalyze reactions. One of these approaches, acid–base catalysis, is of such fundamental importance that it is common to both protein enzymes and RNA-based enzymes, or ribozymes. The hammerhead ribozyme is one such ribozyme that uses an invariant guanine residue as a general base in its catalytic reaction. By changing this to an adenine, we can slow the reaction rate 100,000-fold, permitting us to capture both active, precatalytic, and postcatalytic forms of the ribozyme. We have exploited this approach to obtain near-atomic–resolution three-dimensional structures of the hammerhead ribozyme both before and after catalytic self-cleavage. These structures provide complementary views of the chemical step of hammerhead ribozyme catalysis.