AMINO-ACID-RESIDUES CRITICAL FOR THE INTERACTION BETWEEN BACTERIOPHAGE-T7 DNA-POLYMERASE AND ESCHERICHIA-COLI THIOREDOXIN

Upon infection of Escherichia coil, bacteriophage T7 annexes a host pr otein, thioredoxin, to serve as a processivity factor for its DNA poly merase, T7 gene 5 protein, In a previous communication (Himawan, J., a nd Richardson, C. C. (1992) Proc, Natl. Acad. Sci. U. S. A. 89, 9774-9 778), we reported that an E. coil strain encoding a Gly-74 to Asp-74 ( G74D) thioredoxin mutation could not support wild type T7 growth and t hat in vivo, six mutations in T7 gene 5 could individually suppress th is G74D thioredoxin defect, In the present study, we report the purifi cation and biochemical characterization of the G74D thioredoxin mutant and two suppressor gene 5 proteins, a Glu-319 to Lys-319 (E319K) muta nt of gene 5 protein and an Ala-45 to Thr-45 (A45T) mutant, The suppre ssor E319K mutation, positioned within the DNA polymerization domain o f gene 5 protein, appears to suppress the parental thioredoxin mutatio n by compensating for the binding defect that was caused by the G74D a lteration, We suggest that the Glu-319 residue of T7 gene 5 protein an d the Gly-74 residue of E. coil thioredoxin define a contact point or site of interaction between the two proteins. In contrast, the A45T mu tation in gene 5 protein, located within the 3' to 5' exonuclease doma in, does not suppress the G74D thioredoxin mutation by simple restorat ion of binding affinity, Based upon our understanding of the mechanism s of suppression, we propose a model for the T7 gene 5 protein-E, coil thioredoxin interaction.