Severinov K, Soushko M, Goldfarb A, Nikiforov V
Institute of Molecular Genetics, Russian Academy of Sciences, Moscow.
J Biol Chem. 1993 Jul 15;268(20):14820-5.
Mutations to rifampicin resistance (RifR) in Escherichia coli alter the beta subunit of RNA polymerase (RNAP). A series of new point RifR mutations was isolated with the aid of random polymerase chain reaction-mediated mutagenesis followed by selection on rifampicin (Rif). All of the new RifR mutants, including changes in two novel positions, fell into the two principal clusters previously identified in the middle section of beta. Disruption of the spacer between the two clusters with deletions and insertions led to RNAP that was functional and sensitive to Rif in vitro, indicating that the spacer is not directly involved in Rif binding. However, most of the spacer mutants were strongly resistant to streptolydigin, suggesting that this area is involved in the binding of streptolydigin. An insertion of six consecutive histidines into the spacer was constructed that could be used to anchor RNAP on a Nichelate matrix without the loss of enzymatic activity, indicating that this region is looped out of the RNAP molecule.
大肠杆菌中对利福平耐药（RifR）的突变会改变RNA聚合酶（RNAP）的β亚基。借助随机聚合酶链反应介导的诱变，随后在利福平（Rif）上进行筛选，分离出一系列新的利福平耐药点突变。所有新的RifR突变体，包括两个新位置的变化，都落入了先前在β亚基中间部分鉴定出的两个主要簇中。用缺失和插入破坏两个簇之间的间隔区会导致RNAP在体外具有功能且对利福平敏感，这表明该间隔区不直接参与利福平的结合。然而，大多数间隔区突变体对链霉溶菌素具有很强的抗性，这表明该区域参与链霉溶菌素的结合。构建了一个在间隔区插入六个连续组氨酸的结构，该结构可用于将RNAP锚定在镍螯合基质上而不丧失酶活性，这表明该区域从RNAP分子中环状突出。
