NUCLEOTIDE AND DNA-INDUCED CONFORMATIONAL-CHANGES IN THE BACTERIOPHAGE-T7 GENE-4-PROTEIN

The bacteriophage T7 gene 4 protein is a multifunctional enzyme that h as DNA helicase, primase, and deoxyribonucleotide 5'-triphosphatase ac tivities. Prior studies have shown that in the presence of dTTP or dTD P the gene 4 protein assembles into a functionally active hexamer prio r to binding to single-stranded DNA. In this study, we have examined t he effects of different nucleotide cofactors on the conformation of th e gene 4 protein in the presence and absence of DNA. Gel retardation a nalysis, partial protease digestion, and DNA footprinting all suggest that the gene 4 protein undergoes a conformational change when dTTP is hydrolyzed to dTTP and that in the presence of dTDP the complex with DNA is more open or extended. We have also found that the dissociation constant of the gene 4 protein DNA complex in the presence of dTDP wa s 10-fold lower than that determined in the presence of dTTP, further suggesting that these cofactors exerts different allosteric effects on the DNA-binding site of the gene 4 protein.