Journal
BIOCHEMISTRY
Volume 42, Issue 33, Pages 9924-9936Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi0347757
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- NIGMS NIH HHS [GM62357-01] Funding Source: Medline
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The hammerhead ribozyme is one of the best-studied small RNA enzymes, yet is mechanistically still poorly understood. We measured the Mg2+ dependencies of folding and catalysis for two distinct hammerhead ribozymes, HHL and HHalpha. HHL has three long helical stems and was previously used to characterize Mg2+-induced folding. HHalpha has shorter stems and an A.U tandem next to the cleavage site that increases activity similar to10-fold at 10 mM Mg2+. We find that both ribozymes cleave with fast rates (5-10 min(-1), at pH 8 and 25 degreesC) at nonphysiologically high Mg2+ concentrations, but with distinct Mg2+ dissociation constants for catalysis: 90 mM for HHL and 10 mM for HHa. Using time-resolved fluorescence resonance energy transfer, we measured the stem I-stem II distance distribution as a function of Mg2+ concentration, in the presence and absence of 100 mM Na+, at 4 and 25 degreesC. Our data show two structural transitions. The larger transition (with Mg2+ dissociation constants in the physiological range of similar to1 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 HHalpha 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 Mg2+ is to increase the probability of formation of a properly aligned catalytic core, thus compensating for the absence of naturally occurring kissing-loop interactions.
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