The Study of the Structure and Function of RNA Replicase from RNA Coliphage

为了研究RNA噬菌体RNA复制酶的结构与功能之间的关系，克隆了复制酶<BETA> -亚基蛋白的基因，并通过基因的碱基置换改变了该蛋白中的氨基酸，在体内检测了复制酶的活性。第一年，以Q <BETA>或SP的cDNA克隆构建了携带完整复制酶<BETA> -亚基蛋白基因的质粒，发现每个<BETA> -亚基基因在细胞中均有表达，并可作为正常复制酶亚基发挥作用。去年，利用Q <BETA>的<BETA> -亚基基因，构建了一种突变质粒，该突变质粒将包括RNA噬菌体在内的各种病毒推定的RNA依赖RNA聚合酶中一致片段（tir -Gly- asp - asp）的Gly残基改变为Ala、Ser、Pro、Met或Val，并在体内检测了复制酶的活性。携带插入位点特异性突变复制酶基因的质粒的细胞在体内失去复制酶活性，但通过抑制噬菌体RNA合成来阻止野生型Q <BETA>和SP噬菌体的增殖。然而，Gly残基在另一个位点的取代显示出较少的干扰效应。这些结果表明，在一致序列中引入氨基酸取代会失去核苷酸聚合活性，但仍保留Q <BETA>复制酶的模板识别活性。

To investigate the relationship between the structure and function of RNA replicase of RNA coliphage, gene for the replicase <BETA> -subunit protein was cloned and the replicase activity was examined in vivo after the amino acids in the protein were changed through the base substitutions of the gene. At the first year, the plasmid carrying the intact replicase <BETA> -subunit protein gene was constructed from the cDNA clone of Q <BETA> or SP, and it was found that each <BETA> -subunit gene was expressed and could function as a normal replicase subunit in the cells. At the last year, using the <BETA> -subunit gene of Q <BETA> , the mutant plasmid in which the Gly residue at a consensus segment (Tyr-Gly-Asp-Asp) seen in the putative RNA-dependent RNA polymerases from various viruses including RNA phages has been changed to Ala, Ser, Pro, Met or Val were constructed and the replicase activity was examined in vivo. The cells carrying a plasmid to which the site-specifically mutagenized replicase gene was inserted lost the replicase activity in vivo but prevented the proliferation of the wild-type Q <BETA> and SP phage, by inhibiting the phage RNA synthesis. However, the substitution of the Gly residue at another site showed much less interfering effects. These results suggest that an introduction of amino acid substitutions in the consensus sequence loses the nucleotide polymerizing activity but still retains the template recognition activity of Q <BETA> replicase.