LYS631 RESIDUE IN THE ACTIVE-SITE OF THE BACTERIOPHAGE-T7 RNA-POLYMERASE - AFFINITY LABELING AND SITE-DIRECTED MUTAGENESIS

A highly selective affinity labeling of T7 RNA polymerase with the O-formylphenyl ester of GMP and [alpha-P-32]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.