Rechinsky V O, Kostyuk D A, Lyakhov D L, Chernov B K, Kochetkov S N
V.A. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow.
Mol Gen Genet. 1993 Apr;238(3):455-8. doi: 10.1007/BF00292005.
Random mutagenesis of the gene for bacteriophage T7 RNA polymerase was used to identify functionally essential amino acid residues of the enzyme. A two-plasmid system was developed that permits the straightforward isolation of T7 RNA polymerase mutants that had lost almost all catalytic activity. It was shown that substitutions of Thr and Ala for Pro at the position 563, Ser for Tyr571, Pro for Thr636, Asp for Tyr639 and of Cys for Phe646 resulted in inactivation of the enzyme. It is noteworthy that all these mutations are limited to two short regions that are highly conservative in sequences of monomeric RNA polymerases.
利用噬菌体T7 RNA聚合酶基因的随机诱变来鉴定该酶功能上必需的氨基酸残基。开发了一种双质粒系统，该系统可直接分离几乎丧失所有催化活性的T7 RNA聚合酶突变体。结果表明，在563位用苏氨酸和丙氨酸取代脯氨酸、用丝氨酸取代571位的酪氨酸、用脯氨酸取代636位的苏氨酸、用天冬氨酸取代639位的酪氨酸以及用半胱氨酸取代646位的苯丙氨酸会导致该酶失活。值得注意的是，所有这些突变都局限于两个短区域，这两个区域在单体RNA聚合酶序列中高度保守。
