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-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.
Maksimova
T G
TG
Limnological Institute, Siberian Division of the USSR Academy of Sciences, Irkutsk.
Mustayev
A A
AA
Zaychikov
E F
EF
Lyakhov
D L
DL
Tunitskaya
V L
VL
Akbarov
A Kh
AKh
Luchin
S V
SV
Rechinsky
V O
VO
Chernov
B K
BK
Kochetkov
S N
SN
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
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