The hammerhead ribozyme is one of the best-studied small RNA enzymes, yet is mechanistically still poorly understood. We measured the Mg 2+ dependencies of folding and catalysis for two distinct hammerhead ribozymes, HHL and HHR. HHL has three long helical stems and was previously used to characterize Mg 2+ -induced folding. HHR has shorter stems and an A‚U tandem next to the cleavage site that increases activity ∼10-fold at 10 mM Mg 2+ . We find that both ribozymes cleave with fast rates (5-10 min -1 , at pH 8 and 25°C) at nonphysiologically high Mg 2+ concentrations, but with distinct Mg 2+ dissociation constants for catalysis: 90 mM for HHL and 10 mM for HHR. Using time-resolved fluorescence resonance energy transfer, we measured the stem I-stem II distance distribution as a function of Mg 2+ concentration, in the presence and absence of 100 mM Na + , at 4 and 25°C. Our data show two structural transitions. The larger transition (with Mg 2+ dissociation constants in the physiological range of ∼1 mM, below the catalytic dissociation constants) brings stems I and II close together and is hindered by Na + . The second, globally minor, rearrangement coincides with catalytic activation and is not hindered by Na + . Additionally, the more active HHR exhibits a higher flexibility than HHL under all conditions. Finally, both ribozyme-product complexes have a bimodal stem I-stem II distance distribution, suggesting a fast equilibrium between distinct conformers. We propose that the role of diffusely bound Mg 2+ is to increase the probability of formation of a properly aligned catalytic core, thus compensating for the absence of naturally occurring kissing-loop interactions.Hammerhead ribozymes are found in a number of plant pathogenic viroids and virusoids, where they are involved in self-cleavage and self-ligation of the plus and minus strands during the pathogen's genome replication (1, 2). In addition, the hammerhead ribozyme has been found to mediate self-cleavage of satellite DNA transcripts in the newt (3). Several hammerhead crystal structures have been determined (4-6), and the global structure was confirmed in solution by steady-state fluorescence resonance energy transfer (FRET) 1 (7,8), gel electrophoresis (9), and transient electric birefringence (10) (Figure 1). The ribozyme consists of three helical stems arranged in a "Y-shape", with 11 nucleotides at the junction forming the highly conserved catalytic core (11,12) (Figure 1). The catalytic core is comprised of two domains; stems I and II are arranged in an acute angle by a sharp uridine turn in the backbone trajectory of domain 1, while stems II and III stack coaxially with the formation of two G‚A and one A‚U non-WatsonCrick base pairs in domain 2. The cleavage site is located 3′ to C17, which is buttressed to domain 1 by a single hydrogen bond with C3 (Figure 1). † This work was supported by NIH Grant GM62357-01 to N.G.W., a postdoctoral fellowship from the Swiss National Funds to D.R., a DAAD scholarship to K.W., and a University of Michig...
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