Maksimova T G, Mustayev A A, Zaychikov E F, Lyakhov D L, Tunitskaya V L, Akbarov A Kh, Luchin S V, Rechinsky V O, Chernov B K, Kochetkov S N
Limnological Institute, Siberian Division of the USSR Academy of Sciences, Irkutsk.
Eur J Biochem. 1991 Feb 14;195(3):841-7. doi: 10.1111/j.1432-1033.1991.tb15773.x.
A highly selective affinity labeling of T7 RNA polymerase with the o-formylphenyl ester of GMP and [alpha-32P]UTP was carried out. The site of the labeling was located using limited cleavages with hydroxylamine, bromine, N-chlorosuccinimide and cyanogene bromide and was identified as the Lys631 residue. Site-directed mutagenesis using synthetic oligonucleotides was used to substitute Lys631 by a Gly, Leu or Arg residue. Kinetic studies of the purified mutant enzymes showed alterations of their polymerizing activity. For the Lys----Gly mutant enzyme, anomalous template binding was observed.
使用GMP的邻甲酰苯酯和[α-32P]UTP对T7 RNA聚合酶进行了高度选择性的亲和标记。通过用羟胺、溴、N-氯代琥珀酰亚胺和溴化氰进行有限切割来定位标记位点，并确定其为Lys631残基。使用合成寡核苷酸进行定点诱变，用甘氨酸、亮氨酸或精氨酸残基替代Lys631。对纯化的突变酶的动力学研究表明它们的聚合活性发生了改变。对于Lys→Gly突变酶，观察到异常的模板结合。
