Unravelling the dynamics of RNA degradation by ribonuclease II and its RNA-bound complex

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Abstract
RNA degradation by class II RNase family members plays a fundamental role in the maturation, turnover and quality control of RNA. The crystal structure of an RNase II has now been determined for the first time, in both RNA bound and ligand free forms. Surprisingly, the domain structure does not correspond to that predicted by sequence analysis. The molecule has two points of contact with the RNA: one site anchors the RNA, and the other catalyses cleavage. The structural details explain why RNase II acts only on single-stranded RNA, and how it moves along the RNA to processively degrade it. Determination of the first structure for an RNase II in the absence and presence of RNA reveals that the domain structure is different than sequence analysis had predicted. The structural details explain why RNase II only acts on single-stranded RNA, and how it moves along the RNA to processively degrade it. 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.