Amino acid residues critical for the interaction between bacteriophage T7 DNA polymerase and Escherichia coli thioredoxin.
Upon infection of Escherichia coli, bacteriophage T7 annexes a host protein, thioredoxin, to serve as a processivity factor for its DNA polymerase, T7 gene 5 protein. In a previous communication (Himawan, J., and Richardson, C. C. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9774-9778), we reported that an E. coli strain encoding a Gly-74 to Asp-74 (G74D) thioredoxin mutation could not support wild-type T7 growth and that in vivo, six mutations in T7 gene 5 could individually suppress this G74D thioredoxin defect. In the present study, we report the purification and biochemical characterization of the G74D thioredoxin mutant and two suppressor gene 5 proteins, a Glu-319 to Lys-319 (E319K) mutant of gene 5 protein and an Ala-45 to Thr-45 (A45T) mutant. The suppressor E319K mutation, positioned within the DNA polymerization domain of gene 5 protein, appears to suppress the parental thioredoxin mutation by compensating for the binding defect that was caused by the G74D alteration. We suggest that the Glu-319 residue of T7 gene 5 protein and the Gly-74 residue of E. coli thioredoxin define a contact point or site of interaction between the two proteins. In contrast, the A45T mutation in gene 5 protein, located within the 3' to 5' exonuclease domain, does not suppress the G74D thioredoxin mutation by simple restoration of binding affinity. Based upon our understanding of the mechanisms of suppression, we propose a model for the T7 gene 5 protein-E. coli thioredoxin interaction.
Himawan
J S
JS
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Richardson
C C
CC
D001426:Bacterial Proteins / Q000737:chemistry; D017123:Bacteriophage T7 / Q000201:enzymology* / Q000254:growth & development; D001483:Base Sequence; D004247:DNA / Q000096:biosynthesis; D017931:DNA Primers / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013879:Thioredoxins / Q000737:chemistry*; D014764:Viral Proteins / Q000737:chemistry; D014779:Virus Replication
D001426:Bacterial Proteins; D017931:DNA Primers; D014764:Viral Proteins; D013879:Thioredoxins; D004247:DNA; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase