Capturing hammerhead ribozyme structures in action by modulating general base catalysis.
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 modul...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2008-09-01
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| Series: | PLoS Biology |
| Online Access: | http://europepmc.org/articles/PMC2553840?pdf=render |
| Summary: | 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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| ISSN: | 1544-9173 1545-7885 |