Frazão Carlos, McVey Colin E, Amblar Mónica, Barbas Ana, Vonrhein Clemens, Arraiano Cecília M, Carrondo Maria A
Division of Biological Chemistry, ITQB-Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Apt. 127, 2781-901 Oeiras, Portugal.
Nature. 2006 Sep 7;443(7107):110-4. doi: 10.1038/nature05080.
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 ribonucleases. Ribonuclease II (RNase II) is a major exoribonuclease that intervenes in all of these fundamental processes; it can act independently or as a component of the exosome, an essential RNA-degrading multiprotein complex. 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. Here we report the X-ray crystallographic structures of both the ligand-free (at 2.44 A resolution) and RNA-bound (at 2.74 A 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 alphabeta-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 catalysis. 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.
RNA降解是基因表达调控中的一个决定性因素。RNA的成熟、周转和质量控制由许多不同类型的核糖核酸酶执行。核糖核酸酶II（RNase II）是一种主要的外切核糖核酸酶，参与所有这些基本过程；它可以独立发挥作用，也可以作为外切体的一个组成部分，外切体是一种重要的RNA降解多蛋白复合物。在生命的三个王国中都发现了RNase II样酶，但该家族任何蛋白质的结构数据都没有。在这里，我们报告了无配体（分辨率为2.44埃）和RNA结合（分辨率为2.74埃）形式的大肠杆菌RNase II的X射线晶体结构。与序列预测相反，这些结构表明RNase II被组织成四个结构域：两个冷休克结构域、一个RNB催化结构域（具有前所未有的αβ折叠）和一个S1结构域。该酶在两个不同区域与RNA建立接触，即“锚定”区域和“催化”区域，它们协同作用以提供催化作用。活性位点埋在RNB催化结构域内，位于由四个保守序列基序形成的口袋中。该结构表明催化口袋只对单链RNA可及，并解释了对RNA与DNA切割的特异性。它还通过为RNA易位和酶的持续作用提供结构基础，解释了RNA降解的动态机制。我们提出了一种涉及关键保守残基的外切核糖核酸降解反应机制。我们的三维模型证实了关于RNase II的所有现有生化数据，并阐明了RNA降解的一般基础。此外，它揭示了可以外推到该家族其他成员的重要结构特征。
