Gene 2 protein of bacteriophage T7: purification and requirement for packaging of T7 DNA in vitro.

Abstract

The gene 2 protein of bacteriophage T7 is required for a late stage of T7 DNA replication because T7 gene 2 mutants fail to form normal concatemeric structures during the processing of newly synthesized T7 DNA. Extracts of gene 2 mutant phage-infected cells are unable to package T7 DNA into phage heads to form viable phage, as determined by an in vitro packaging assay for T7 DNA. Packaging activity can be stimulated greater than 100-fold in mutant extracts by the addition of extract prepared from cells infected with phage carrying a wild-type T7 gene 2, thus providing a complementation assay for the gene 2 protein. With this assay, the gene 2 protein has been purified to approximately 50% homogeneity. Purified preparations of the protein inhibit the activity of Escherichia coli RNA polymerase but have little effect on the activity of T7 RNA polymerase but have little effect on the activity of T7 RNA polymerase. The requirement for the gene 2 protein during T7 DNA replication may involve inactivation of E. coli RNA polymerase because the antibiotic rifampicin, a specific inhibitor of E. coli RNA polymerase, can substitute for the gene 2 protein in the in vitro packaging assay.