Abstract
RNA degradation is a determining factor in the control of gene expression. The maturation, turnover and quality control of RNA is performed by many different classes of ribonucleases1. Ribonuclease II (RNase II) is a major exoribonuclease that intervenes in all of these fundamental processes1; it can act independently or as a component of the exosome, an essential RNA-degrading multiprotein complex2. RNase II-like enzymes are found in all three kingdoms of life, but there are no structural data for any of the proteins of this family1,2,3,4,5. Here we report the X-ray crystallographic structures of both the ligand-free (at 2.44 Å resolution) and RNA-bound (at 2.74 Å resolution) forms of Escherichia coli RNase II. In contrast to sequence predictions, the structures show that RNase II is organized into four domains: two cold-shock domains, one RNB catalytic domain, which has an unprecedented αβ-fold, and one S1 domain. The enzyme establishes contacts with RNA in two distinct regions, the ‘anchor’ and the ‘catalytic’ regions, which act synergistically to provide catalysis6. The active site is buried within the RNB catalytic domain, in a pocket formed by four conserved sequence motifs. The structure shows that the catalytic pocket is only accessible to single-stranded RNA, and explains the specificity for RNA versus DNA cleavage. It also explains the dynamic mechanism of RNA degradation by providing the structural basis for RNA translocation and enzyme processivity. We propose a reaction mechanism for exonucleolytic RNA degradation involving key conserved residues. Our three-dimensional model corroborates all existing biochemical data for RNase II, and elucidates the general basis for RNA degradation. Moreover, it reveals important structural features that can be extrapolated to other members of this family.
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Acknowledgements
We thank L. Maquat, C. Condon and P. Lindley for critical reading of the manuscript; R. Parker, W. Hendrickson and T. Blundell for advice and encouragement; and the staff of beamlines D14-1/ID14-2/ID13 from ESRF, Grenoble, for data collection technical support. This work was supported by Fundação para a Ciência e Tecnologia, Portugal. Author Contributions C.F., C.E.M. (crystallography) and M.A. (molecular biology) contributed equally to this work.
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Diffraction data and atomic coordinates of RNase II and its D209N-RNA bound mutant complex have been deposited in the Protein Data Bank with accession numbers r2ix0sf and 2ix0, and r2ix1sf and 2ix1, respectively. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.
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Supplementary Data
This file contains Supplementary Methods, Supplementary Tables 1–5, Supplementary Figures 1–11 and Supplementary references. (PDF 1486 kb)
Supplementary Movie 1
This movie shows a global view of the 13 nucleotides RNA molecule bound to RNase II D209N mutant, with RNA in sticks coloured from blue to red according to their growing atomic displacement parameters, and with the enzyme cartoon coloured by domains, namely CSD1 in yellow, CSD2 in orange, RNB in cyan and S1 in green. (MPG 6702 kb)
Supplementary Movie 2
This movie shows a global view of the 13 nucleotides RNA molecule bound to RNase II D209N mutant, with RNA in sticks coloured from blue to red according to their growing atomic displacement This movie shows zoomed views of the interactions between RNase II and the bound RNA molecule, namely at the anchor region, through the flexible intermediate nucleotides, and at the 5 terminal RNA nucleotides clamped between conserved aromatic residues, in the catalytic cavity. (MOV 6291 kb)
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Frazão, C., McVey, C., Amblar, M. et al. Unravelling the dynamics of RNA degradation by ribonuclease II and its RNA-bound complex. Nature 443, 110–114 (2006). https://doi.org/10.1038/nature05080
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DOI: https://doi.org/10.1038/nature05080
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