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 en zyme. A two-plasmid system was developed that permits the straightforw ard isolation of T7 RNA polymerase mutants that had lost almost all ca talytic activity. It was shown that substitutions of Thr and Ala for P ro 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 n oteworthy that all these mutations are limited to two short regions th at are highly conservative in sequences of monomeric RNA polymerases.