title: Diffusely bound Mg2+ ions slightly reorient stems I and II of the hammerhead ribozyme to increase the probability of formation of the catalytic core.
abstract: 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 HH alpha. HHL has three long helical stems and was previously used to characterize Mg(2+)-induced folding. HH alpha has shorter stems and an A.U tandem next to the cleavage site that increases activity approximately 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 degrees C) at nonphysiologically high Mg(2+) concentrations, but with distinct Mg(2+) dissociation constants for catalysis: 90 mM for HHL and 10 mM for HH alpha. 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 degrees C. Our data show two structural transitions. The larger transition (with Mg(2+) dissociation constants in the physiological range of approximately 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 HH alpha 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.
authors.affiliation: Department of Chemistry and Biophysics Research Division, The University of Michigan, 930 North University Avenue, Ann Arbor, Michigan 48109-1055, USA.
authors.forename: David
authors.initials: D
authors.lastname: Rueda
authors.forename: Katrin
authors.initials: K
authors.lastname: Wick
authors.forename: S Elizabeth
authors.initials: SE
authors.lastname: McDowell
authors.forename: Nils G
authors.initials: NG
authors.lastname: Walter
mesh_terms: D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry*; D013329:Structure-Activity Relationship
chemical_list: D016337:RNA, Catalytic; D012313:RNA; D008274:Magnesium