Lys631 residue in the active site of the bacteriophage T7 RNA polymerase. Affinity labeling and site-directed mutagenesis

Open Access
Publisher Website
Google Scholar
Abstract
A highly selective affinity labeling of T7 RNA polymerase with the o‐formylphenyl ester of GMP and [α‐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.