1. Division of Biological Chemistry,
2. Division of Biology, ITQB–Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Apt. 127, 2781-901 Oeiras, Portugal
3. Global Phasing Limited, Sheraton House, Castle Park, Cambridge CB3 0AX, UK
4. These authors contributed equally to this work

Correspondence to: Cecília M. Arraiano²Maria A. Carrondo¹ Correspondence and requests for materials should be addressed to M.A.C (Email: carrondo@itqb.unl.pt) or C.M.A (Email: cecilia@itqb.unl.pt). 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.

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 family^{1, 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 -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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