Kawakami J, Kumar P K, Suh Y A, Nishikawa F, Kawakami K, Taira K, Ohtsuka E, Nishikawa S
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Eur J Biochem. 1993 Oct 1;217(1):29-36. doi: 10.1111/j.1432-1033.1993.tb18214.x.
Models for the secondary structure of genomic and antigenomic self-cleaving RNAs of human hepatitis delta (delta) virus (HDV) have been proposed by several groups. Our recent results support a pseudoknot structure and have allowed us to identify functionally important nucleotides in single-stranded regions [nucleotides 726-731 (SSrA) and nucleotides 762-766 (SSrB)]. For the identification of the important residues in the remaining single-stranded region, nucleotides 708-715 (SSrC), of the genomic HDV ribozyme, we made derivatives with a single-base substitution in the SSrC region. To screen inactive mutants rapidly, we use a simplified in-vitro selection method. Among the various base substitutions in mutants in the SSrC, U708A, C709(A/G/U) and G713C variants had less than 10% of the cleavage activity of the wild-type SSrC (HDV86). By analyzing the self-cleavage activities of various mutants, we determined the base requirements for SSrC as 5'-(U/C/G)-C-N-N-(C/A/G)-(G/A/U)-N-N-3'.
几个研究小组已经提出了人类丁型肝炎病毒（HDV）基因组和反基因组自我切割RNA二级结构的模型。我们最近的研究结果支持假结结构，并使我们能够识别单链区域中功能重要的核苷酸[核苷酸726 - 731（SSrA）和核苷酸762 - 766（SSrB）]。为了识别基因组HDV核酶剩余单链区域（核苷酸708 - 715，SSrC）中的重要残基，我们构建了在SSrC区域有单碱基替换的衍生物。为了快速筛选无活性突变体，我们使用了一种简化的体外筛选方法。在SSrC区域突变体的各种碱基替换中，U708A、C709（A / G / U）和G713C变体的切割活性不到野生型SSrC（HDV86）的10%。通过分析各种突变体的自我切割活性，我们确定了SSrC的碱基需求为5' -（U / C / G）- C - N - N -（C / A / G）-（G / A / U）- N - N - 3'。
