Abstract
The hairpin ribozyme catalyses sequence-specific cleavage of RNA. The active site of this natural RNA results from the docking of two irregular helices: stems A and B. One strand of stem A harbours the scissile bond. The 2.4 Å resolution structure of a hairpin ribozyme–inhibitor complex reveals that the ribozyme aligns the 2′-OH nucleophile and the 5′-oxo leaving group by twisting apart the nucleotides that flank the scissile phosphate. The base of the nucleotide preceding the cleavage site is stacked within stem A; the next nucleotide, a conserved guanine, is extruded from stem A and accommodated by a highly complementary pocket in the minor groove of stem B. Metal ions are absent from the active site. The bases of four conserved purines are positioned potentially to serve as acid-base catalysts. This is the first structure determination of a fully assembled ribozyme active site that catalyses a phosphodiester cleavage without recourse to metal ions.
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Acknowledgements
We thank T. Earnest, L. Hung and G. McDermott for help at ALS beamline 5.0.2; C. Hoang for biochemical support; J. Bolduc, P. Heath and B. Shen for computational and crystallographic support; K. Nagai for U1A plasmids; and S. Biggins, M. Holmes, P. Li, M. Rosenberg, S. Ryder, S. Sigurdsson, B. Stoddard, S. Strobel, R. Strong, G. Varani, D. Wilson and K. Zhang for discussions. This work was supported by institutional funds from the Fred Hutchinson Cancer Research Center (FHCRC). Access to ALS beamline 5.0.2 as part of the principal research consortium was made possible by general support from the FHCRC. P.B.R. is a post-doctoral trainee of the Chromosome Metabolism and Cancer training grant from the National Cancer Institute to the FHCRC.
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Figure 1.
Single turnover cleavage assay. End-labeled substrate RNA (200 nM) was incubated with ribozyme (1 m M) in reaction buffer (1 mM MgCl2, and 50 mM Tris pH 7.5) at 25°C for the times indicated. Reactions were resolved on a 20% polyacrylamide, 8M urea gel. The substrate RNA has the same sequence as the inhibitor strand used in the crystals but is all-ribose. It was incubated with either a wild-type four-helix junction ribozyme, or the crystallization construct with and without U1A-RBD. Reactions do not go to completion because the ribozymes also catalyze ligation.
Figure 2.
Stereoview of a portion of the 2.4 쎅 resolution ‘solvent-flattened’ experimental electron density map represented as blue and red mesh. The contour levels are one and three s.d. above the mean peak height, respectively. Superimposed on the density is the refined model of the active site oriented as in Fig. 5d.
Figure 3.
Stereoview of a portion of the sA-weighted simulated-annealing omit 2|Fo| -|Fc| electron density map. The contour levels, colors, and superimposed model are the same as in the previous figure.
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Rupert, P., Ferré-D'Amaré, A. Crystal structure of a hairpin ribozyme–inhibitor complex with implications for catalysis. Nature 410, 780–786 (2001). https://doi.org/10.1038/35071009
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DOI: https://doi.org/10.1038/35071009
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