Nishikawa F, Nishikawa S
National Institute of Bioscience and Human Technology, AIST, MITI, 1-1 Higashi, Tsukuba Science City, Ibaraki 305-8566, Japan.
Nucleic Acids Res. 2000 Feb 15;28(4):925-31. doi: 10.1093/nar/28.4.925.
The tertiary structure of the 3'-cleaved product of the genomic hepatitis delta virus (HDV) ribozyme was solved by X-ray crystallographic analysis. In this structure, three single-stranded regions (SSrA, -B and -C) interact intricately with one another via hydrogen bonds between nucleotide bases, phosphate oxygens and 2'-OHs to form a nested double pseudoknot structure. Among these interactions, two Watson-Crick (W-C) base pairs, 726G-710C and 727G-709C, that form between SSrA and SSrC (P1.1) seem to be especially important for compact folding. To characterize the importance of these base pairs, ribozymes were subjected to in vitro selection from a pool of RNA molecules randomly substituted at positions 709, 710, 726 and 727. The results establish the importance of the two W-C base pairs for activity, although some mutants are active with one G-C base pair. In addition, the kinetic parameters were analyzed in all 16 combinations with two canonical base pairs. Comparison of variant ribozymes with the wild-type ribozyme reveals that the difference in reaction rates for these variants (DeltaDelta G (double dagger)) is not simply accounted for by the differences in the stability of P1.1 (DeltaDelta G (0)(37)). The role played by Mg(2+)ions in formation of the P1.1 structure is also discussed.
通过X射线晶体学分析解析了基因组丁型肝炎病毒（HDV）核酶3'切割产物的三级结构。在该结构中，三个单链区域（SSrA、-B和-C）通过核苷酸碱基、磷酸氧和2'-OH之间的氢键相互复杂地作用，形成嵌套的双假结结构。在这些相互作用中，SSrA和SSrC（P1.1）之间形成的两个沃森-克里克（W-C）碱基对，即726G-710C和727G-709C，似乎对紧密折叠尤为重要。为了表征这些碱基对的重要性，从在709、710、726和727位随机取代的RNA分子库中对核酶进行体外筛选。结果确定了这两个W-C碱基对对于活性的重要性，尽管一些突变体在只有一个G-C碱基对时仍具有活性。此外，对具有两个标准碱基对的所有16种组合的动力学参数进行了分析。将变体核酶与野生型核酶进行比较表明，这些变体的反应速率差异（ΔΔG‡）不能简单地由P1.1的稳定性差异（ΔΔG(0)(37)）来解释。还讨论了Mg(2+)离子在P1.1结构形成中所起的作用。
