THE HISTIDINE-805 IN MOTIF-C OF THE PHAGE SP6 RNA-POLYMERASE IS ESSENTIAL FOR ITS ACTIVITY AS REVEALED BY RANDOM MUTAGENESIS

In order to identify functional residues of the bacteriophage SP6 RNA polymerase, its C-terminal one-twelfth region was randomly mutagenized using polymerase chain reactions of its gene under the conditions for reduced fidelity of Taq DNA polymerase. Using a two-vector system tha t permits phenotypic isolation of mutants with reduced in vivo transcr iption activity, 3 single and 1 multiple mutants were isolated. A sing le substitution of Gln for His805 resulted in complete inactivation of the enzyme. A multiple mutant carrying substitutions at 808, 820, 835 , 843 and 848 also abolished the activity. However, changes of Pro856- ->Ser and Asp862-->Glu individually reduced the activity only slightly . It is noteworthy that His805 is one of the two motif-C residues that are absolutely conserved among all the DNA polymerases and monomeric RNA polymerases.