Lys631 residue in the active site of the bacteriophage T7 RNA polymerase. Affinity labeling and site-directed mutagenesis. |
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| Authors: | T G Maksimova A A Mustayev E F Zaychikov D L Lyakhov V L Tunitskaya A Kh Akbarov S V Luchin V O Rechinsky B K Chernov S N Kochetkov |
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| Institution: | Limnological Institute, Siberian Division of the USSR Academy of Sciences, Irkutsk. |
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| Abstract: | 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. |
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