title: 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.
abstract: 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.
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D009154:Mutation; D010084:Oxidation-Reduction; D013604:T-Phages / Q000235:genetics / Q000378:metabolism*; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D014764:Viral Proteins; D013879:Thioredoxins; D004259:DNA-Directed DNA Polymerase