Deoxyribonucleic acid polymerase of bacteriophage T7. Purification and properties of the phage-encoded subunit, the gene 5 protein

J Biol Chem. 1979 Nov 25;254(22):11591-7.


DNA polymerase of bacteriophage T7 is composed of two subunits, the gene 5 protein of the phage and the host-specified thioredoxin. The gene 5 protein has been purified 7400-fold to homogeneity from bacteriophage T7-infected Escherichia coli 7400 trxA cells that lack thioredoxin. The purification procedure has been monitored by using a complementation assay in which thioredoxin interacts with the gene 5 protein to form an active DNA polymerase. The purified gene 5 protein is a single polypeptide having a molecular weight of 87,000. The gene 5 protein itself has only 1 to 2% of the polymerase activity of T7 DNA polymerase. However, T7 DNA polymerase can be reconstituted by the addition of homogeneous thioredoxin to the gene 5 protein. Optimal reconstitution is obtained when the molar ratio of thioredoxin/gene 5 protein is 150. Under these conditions, the gene 5 protein attains approximately 80% of the activity of an equal amount of T7 DNA polymerase. The apparent Km for thioredoxin in the reaction to restore DNA polymerase activity is 2.8 x 10(-8) M. The enzymatic properties of the reconstituted enzyme are indistinguishable from those of T7 DNA polymerase synthesized in vivo; the reconstituted polymerase interacts with T7 gene 4 protein to catalyze DNA synthesis on duplex DNA templates.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • DNA-Directed DNA Polymerase / isolation & purification
  • DNA-Directed DNA Polymerase / metabolism*
  • Escherichia coli / enzymology*
  • Genes, Viral*
  • Genetic Complementation Test
  • Kinetics
  • Macromolecular Substances
  • Molecular Weight
  • T-Phages / enzymology*
  • Thioredoxins / pharmacology
  • Viral Proteins / isolation & purification
  • Viral Proteins / metabolism*


  • Macromolecular Substances
  • Viral Proteins
  • Thioredoxins
  • DNA-Directed DNA Polymerase