大肠杆菌严谨型RNA聚合酶的筛选及体内转录活性测定

Stringent RNA polymerase of E. coli and its in vivo transcriptional activity

摘要:

利用选择性培养基筛选大肠杆菌自然突变菌株,经噬菌体P1转导和蛋白质互补试验,发现一株突变体(LCH001)的突变基因发生在编码RNA聚合酶β′亚基的rpoC基因上,经DNA序列分析,发现突变位点发生在第3406个碱基上,由G变成了T,导致编码的氨基酸由甘氨酸(GGT)变成半胱氨酸(TGT)。体内转录试验表明该突变RNA聚合酶转录严谨型启动子控制基因的活性显著降低,其β-半乳糖苷酶的活性是野生型菌株的18%,而转录非严谨型启动子控制基因的活性显著提高,其β-半乳糖苷酶的活性约是野生型菌株的5倍。研究结果对探讨RNA聚合酶结构与功能的关系以及RNA聚合酶在细菌严谨反应过程中的作用具有重要意义。

Abstract:

Several spontaneous E. coli mutants with the similar phenotype as that in the condition of amino acid deficiency were obtained on the selective media. One of the mutants (LCH001) showing slow growth phenotype on LB agar plate and pink or white colonies on MacConkey agar plate was mapped at rpoC gene encoding the β′ subunit of RNA polymerase by phage P1 transduction and transformation assays and found to be a new site mutation from G to T at 3406bp in the rpoC gene, which resulted in the amino acid change from Glycine (GGT) to Cysteine (TGT). The effect of the mutation on transcriptional activity of both stringent and non-stringent controlled promoters in vivo was measured by determining the β-galactolactase activity of the growing cells. Results showed that the transcriptional activity of the mutant LCH001 reduced greatly on the stringent promoter, but increased significantly on the non-stringent promoter. The β-galactolactase activity of the mutant LCH001 transcribed on stringent promoter was 18% lower, but 5-fold higher on the non-stringent controlled promoter than that of the wild-type strain CLT5034. This finding may give insights into future studies of the structure-function relationship of RNA polymerase as well as its role in the stringent response of bacteria.