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.
MeSH terms
-
Affinity Labels / metabolism
-
Amino Acid Sequence
-
Base Sequence
-
Binding Sites
-
DNA-Directed RNA Polymerases / genetics
-
DNA-Directed RNA Polymerases / metabolism*
-
Escherichia coli / enzymology*
-
Guanosine Monophosphate / analogs & derivatives*
-
Guanosine Monophosphate / metabolism
-
Hydroxylamine
-
Hydroxylamines / pharmacology
-
Kinetics
-
Lysine*
-
Molecular Sequence Data
-
Mutagenesis, Site-Directed*
-
Oligonucleotide Probes
-
Plasmids
-
Restriction Mapping
-
Sequence Homology, Nucleic Acid
-
T-Phages / enzymology*
-
T-Phages / genetics
Substances
-
Affinity Labels
-
Hydroxylamines
-
Oligonucleotide Probes
-
2-formylphenyl guanosine monophosphate ester
-
Hydroxylamine
-
Guanosine Monophosphate
-
DNA-Directed RNA Polymerases
-
Lysine