Random mutagenesis of the gene for bacteriophage T7 RNA polymerase.
Random mutagenesis of the gene for bacteriophage T7 RNA polymerase was used to identify functionally essential amino acid residues of the enzyme. A two-plasmid system was developed that permits the straightforward isolation of T7 RNA polymerase mutants that had lost almost all catalytic activity. It was shown that substitutions of Thr and Ala for Pro at the position 563, Ser for Tyr571, Pro for Thr636, Asp for Tyr639 and of Cys for Phe646 resulted in inactivation of the enzyme. It is noteworthy that all these mutations are limited to two short regions that are highly conservative in sequences of monomeric RNA polymerases.
V.A. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow.
V O
VO
Rechinsky
D A
DA
Kostyuk
D L
DL
Lyakhov
B K
BK
Chernov
S N
SN
Kochetkov
D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D012321:DNA-Directed RNA Polymerases