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. © 2008 Chi et al.
CITATION STYLE
Chi, Y. I., Martick, M., Lares, M., Kim, R., Scott, W. G., & Kim, S. H. (2008). Capturing hammerhead ribozyme structures in action by modulating general base catalysis. PLoS Biology, 6(9), 2060–2068. https://doi.org/10.1371/journal.pbio.0060234
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