Capturing hammerhead ribozyme structures in action by modulating general base catalysis

Young In Chi, Monika Martick, Monica Lares, Rosalind Kim, William G. Scott, Sung Hou Kim

Research output: Contribution to journalArticlepeer-review

67 Scopus citations

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.

Original languageEnglish (US)
Article numbere234
Pages (from-to)2060-2068
Number of pages9
JournalPLoS biology
Volume6
Issue number9
DOIs
StatePublished - Sep 2008

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