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.
Highlights
The hammerhead ribozyme, since its discovery in satellite virus RNA genomes [1,2], has been a central focus of experiments designed to correlate RNA structure with RNA catalysis, as it is a comparatively small RNA whose biochemistry has been intensively investigated using a wide variety of approaches [3,4,5]
Where multiple values are reported, the number in parentheses corresponds to the highest resolution shell, whereas numbers not residing within parentheses correspond to overall crystallographic statistics
Concluding Remarks Until 2003, it was not recognized that a tertiary contact region possessing little recognizable sequence conservation is critical for optimal catalysis [6,7], and subsequently, it was Refinement Cross-validation method Free R value test set selection R value Free R value Free R value test set size (%) Free R value test set count Number of non-hydrogen atoms Mean B value Estimated overall coordinate error
Summary
The hammerhead ribozyme, since its discovery in satellite virus RNA genomes [1,2], has been a central focus of experiments designed to correlate RNA structure with RNA catalysis, as it is a comparatively small RNA whose biochemistry has been intensively investigated using a wide variety of approaches [3,4,5]. The discovery that natural hammerhead RNAs having tertiary contacts distant from the active site may enhance catalysis up to approximately 1,000-fold relative to ‘‘minimal’’ hammerheads [6,7,8,9] compelled renewed mechanistic and structural investigations. The most well-characterized member of the first class of natural hammerheads occurs within the satellite RNA of the tobacco ringspot virus (sTRSV), which is the first hammerhead ribozyme discovered [10]. The structure [13] of a full-length Schistosome hammerhead [12] ribozyme-competitive inhibitor complex in which a substrate analog having a modified 29-OMeC17 nucleophile was recently obtained, revealing how G12 becomes positioned to initiate cleavage as a general base, and how G8 may function as a general acid in hammerhead ribozyme catalysis. The substrate was inactivated by replacing the nucleophilic 29OH of the cleavage-site nucleotide (C17) with an inert ether linkage, potentially altering the active site environment
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