Capturing hammerhead ribozyme structures in action by modulating general base catalysis |
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Authors: | Chi Young-In Martick Monika Lares Monica Kim Rosalind Scott William G Kim Sung-Hou |
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Institution: | Department of Molecular and Cellular Biochemistry, Center for Structural Biology, University of Kentucky, Lexington, Kentucky, United States of America. ychi@uky.edu |
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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 pKa 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. |
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