Interaction of mutant thioredoxins of Escherichia coli with the gene 5 protein of phage T7. The redox capacity of thioredoxin is not required for stimulation of DNA polymerase activity

J Biol Chem. 1986 Nov 15;261(32):15006-12.


DNA polymerase activity in Escherichia coli cells infected with bacteriophage T7 resides in a protein complex consisting of the T7 gene 5 protein and E. coli thioredoxin in a 1 to 1 stoichiometry. We have analyzed nine mutant thioredoxins, both in vivo and in vitro, for their ability to interact with the T7 gene 5 protein and stimulate the DNA polymerase and exonuclease activities inherent in gene 5 protein. The efficiency of plating of T7 on E. coli thioredoxin mutants depends strongly on the copy number of the respective mutant thioredoxin allele. Plating efficiencies at a constant copy number correlate well with the affinity of the purified mutant proteins for T7 gene 5 protein. The observed dissociation constant, Kobs, is increased between 5 and several hundredfold at 42 degrees C compared to wild-type thioredoxin. The maximum polymerase activity of the reconstituted gene 5 protein-thioredoxin complex at saturating concentrations of mutant thioredoxins, however, is reduced by less than 20%. Consequently, none of the mutant thioredoxins acts as a competitive inhibitor of wild-type thioredoxin. The active-site disulfide of thioredoxin is not essential for the activities of the gene 5 protein-thioredoxin complex. Both cysteines can be replaced without significantly affecting the maximum polymerase or exonuclease activities. Substitution or alkylation of either cysteine, however, reduces the affinity for gene 5 protein drastically, indicating that the active site is part of the thioredoxin surface involved in the protein-protein interaction.

Publication types

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

MeSH terms

  • Bacterial Proteins / metabolism*
  • DNA-Directed DNA Polymerase / metabolism*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Kinetics
  • Mutation*
  • Oxidation-Reduction
  • T-Phages / genetics
  • T-Phages / metabolism*
  • Thioredoxins / genetics
  • Thioredoxins / metabolism*
  • Viral Proteins / genetics
  • Viral Proteins / metabolism*


  • Bacterial Proteins
  • Viral Proteins
  • Thioredoxins
  • DNA-Directed DNA Polymerase