Kumar P K, Suh Y A, Miyashiro H, Nishikawa F, Kawakami J, Taira K, Nishikawa S
Department of Cellular and Molecular Biology, Agency of Industrial Science & Technology, Ibaraki, Japan.
Nucleic Acids Res. 1992 Aug 11;20(15):3919-24. doi: 10.1093/nar/20.15.3919.
In elucidating function of two important single-stranded regions [SSrA (726-731 nt) and SSrB (762-766 nt)] derived mainly from three secondary structure models in genomic hepatitis delta virus (HDV) ribozyme possessing self-cleavage activity, we have constructed several random mutants at those two regions on the HDV88 molecule (683-770 nt) by oligonucleotide-directed mutagenesis. When self-cleavage activities were compared among mutants, at the region SSrA, G726 was found to play an important role during cleavage reaction since substitutions of the base to A (mutant A20) or C (mutant A16) or U (mutant A23), reduced the ribozyme activity to very low levels suggesting the importance of G726 position. C763 at SSrB region was found to play a more significant role during catalysis than G726 (at region SSrA) since any substitutions at C763 completely inactivated the ribozyme. Other bases located in these two regions could be substituted to other bases at the expense of some self-cleavage activity. The results presented here together with our previous deletion analysis indicate that these two regions may play an important role during cleavage process.
在阐明主要源自基因组丁型肝炎病毒（HDV）核酶的三种二级结构模型、具有自我切割活性的两个重要单链区域[SSrA（726 - 731核苷酸）和SSrB（762 - 766核苷酸）]的功能时，我们通过寡核苷酸定向诱变在HDV88分子（683 - 770核苷酸）的这两个区域构建了几个随机突变体。当比较突变体之间的自我切割活性时，在SSrA区域，发现G726在切割反应中起重要作用，因为将该碱基替换为A（突变体A20）或C（突变体A16）或U（突变体A23）会使核酶活性降低到非常低的水平，这表明G726位置很重要。发现SSrB区域的C763在催化过程中比G726（在SSrA区域）起更重要的作用，因为C763处的任何替换都会使核酶完全失活。这两个区域中的其他碱基可以被替换为其他碱基，但会牺牲一些自我切割活性。此处呈现的结果以及我们之前的缺失分析表明，这两个区域可能在切割过程中起重要作用。
