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

为了研究RNA大肠杆菌噬菌体RNA复制酶的结构与功能的关系，克隆了复制酶<BETA>亚基蛋白基因，并通过碱基替换改变蛋白质中的氨基酸，在体内检测了复制酶的活性。在第一年，<BETA>从Q或SP的cDNA克隆中构建了携带完整复制酶-亚基蛋白基因的质粒<BETA>，发现每个<BETA>-亚基基因在细胞中表达并可作为正常复制酶亚基发挥功能。去年，利用<BETA>Q的α亚基基因<BETA>，构建了突变质粒，其中在各种病毒包括RNA聚合酶的推定RNA依赖性RNA聚合酶中所见的共有片段（Tyr-Gly-Asp-Asp）处的Gly残基已被改变为Ala、Ser、Pro、Met或瓦尔，并在体内检测了复制酶活性。携带插入了位点特异性诱变的复制酶基因的质粒的细胞在体内失去了复制酶活性，但<BETA>通过抑制噬菌体RNA合成而阻止了野生型Q和SP噬菌体的增殖。然而，在另一个位点的甘氨酸残基的取代显示出小得多的干扰效果。这些结果表明，在共有序列中引入氨基酸取代失去了核苷酸聚合活性，但仍保留了Q复制酶的模板识别活性<BETA>。

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.