Capturing hammerhead ribozyme structures in action by modulating general base catalysis

  • Chi Y
  • Martick M
  • Lares M
 et al. 
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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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Authors

  • Young In Chi

  • Monika Martick

  • Monica Lares

  • Rosalind Kim

  • William G. Scott

  • Sung Hou Kim

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