title: Lys631 residue in the active site of the bacteriophage T7 RNA polymerase. Affinity labeling and site-directed mutagenesis.
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.
authors.affiliation: Limnological Institute, Siberian Division of the USSR Academy of Sciences, Irkutsk.
authors.forename: T G
authors.initials: TG
authors.lastname: Maksimova
authors.forename: A A
authors.initials: AA
authors.lastname: Mustayev
authors.forename: E F
authors.initials: EF
authors.lastname: Zaychikov
authors.forename: D L
authors.initials: DL
authors.lastname: Lyakhov
authors.forename: V L
authors.initials: VL
authors.lastname: Tunitskaya
authors.forename: A Kh
authors.initials: AKh
authors.lastname: Akbarov
authors.forename: S V
authors.initials: SV
authors.lastname: Luchin
authors.forename: V O
authors.initials: VO
authors.lastname: Rechinsky
authors.forename: B K
authors.initials: BK
authors.lastname: Chernov
authors.forename: S N
authors.initials: SN
authors.lastname: Kochetkov
mesh_terms: D000345:Affinity Labels / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D006157:Guanosine Monophosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007700:Kinetics; D008239:Lysine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010957:Plasmids; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013604:T-Phages / Q000201:enzymology* / Q000235:genetics
chemical_list: D000345:Affinity Labels; D006898:Hydroxylamines; D015345:Oligonucleotide Probes; C067608:2-formylphenyl guanosine monophosphate ester; D019811:Hydroxylamine; D006157:Guanosine Monophosphate; D012321:DNA-Directed RNA Polymerases; D008239:Lysine