Kapral Gary J, Jain Swati, Noeske Jonas, Doudna Jennifer A, Richardson David C, Richardson Jane S
Department of Biochemistry, Duke University, Durham, NC 27710, USA.
Department of Biochemistry, Duke University, Durham, NC 27710, USA Program in Computational Biology and Bioinformatics, Duke University, Durham, NC 27710, USA.
Nucleic Acids Res. 2014 Nov 10;42(20):12833-46. doi: 10.1093/nar/gku992. Epub 2014 Oct 17.
The hepatitis delta virus (HDV) ribozyme is a self-cleaving RNA enzyme essential for processing viral transcripts during rolling circle viral replication. The first crystal structure of the cleaved ribozyme was solved in 1998, followed by structures of uncleaved, mutant-inhibited and ion-complexed forms. Recently, methods have been developed that make the task of modeling RNA structure and dynamics significantly easier and more reliable. We have used ERRASER and PHENIX to rebuild and re-refine the cleaved and cis-acting C75U-inhibited structures of the HDV ribozyme. The results correct local conformations and identify alternates for RNA residues, many in functionally important regions, leading to improved R values and model validation statistics for both structures. We compare the rebuilt structures to a higher resolution, trans-acting deoxy-inhibited structure of the ribozyme, and conclude that although both inhibited structures are consistent with the currently accepted hammerhead-like mechanism of cleavage, they do not add direct structural evidence to the biochemical and modeling data. However, the rebuilt structures (PDBs: 4PR6, 4PRF) provide a more robust starting point for research on the dynamics and catalytic mechanism of the HDV ribozyme and demonstrate the power of new techniques to make significant improvements in RNA structures that impact biologically relevant conclusions.
丁型肝炎病毒（HDV）核酶是一种自我切割的RNA酶，在滚环式病毒复制过程中对病毒转录本的加工至关重要。1998年解析出了切割后核酶的首个晶体结构，随后又有未切割、突变抑制和离子复合形式的结构。最近，已开发出一些方法，使RNA结构和动力学建模任务显著变得更容易且更可靠。我们使用ERRASER和PHENIX对HDV核酶的切割型和顺式作用C75U抑制型结构进行了重建和重新精修。结果校正了局部构象，并确定了RNA残基的替代构象，其中许多位于功能重要区域，从而提高了两种结构的R值和模型验证统计数据。我们将重建后的结构与分辨率更高的核酶反式作用脱氧抑制型结构进行比较，得出结论：虽然两种抑制型结构都与当前公认的锤头状切割机制一致，但它们并未为生化和建模数据增添直接的结构证据。然而，重建后的结构（蛋白质数据银行编号：4PR6、4PRF）为HDV核酶的动力学和催化机制研究提供了一个更可靠的起点，并证明了新技术在显著改进影响生物学相关结论的RNA结构方面的强大作用。
