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The histidine‐805 in motif‐C of the phage SP6 RNA polymerase is essential for its activity as revealed by random mutagenesis
Author(s) -
Jeong Woojin,
Kang Changwon
Publication year - 1997
Publication title -
iubmb life
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.132
H-Index - 113
eISSN - 1521-6551
pISSN - 1521-6543
DOI - 10.1080/15216549700203131
Subject(s) - motif (music) , polymerase , histidine , mutagenesis , chemistry , microbiology and biotechnology , biology , genetics , dna , gene , mutation , amino acid , physics , acoustics
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 that permits phenotypic isolation of mutants with reduced in vivo transcription activity, 3 single and 1 multiple mutants were isolated. A single 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.