A bacteriophage T 7 RNA polymerase / promoter system for controlled exclusive expression of specific genes ( T 7 DNA polymerase / T 7 gene 5 protein / proteolysis / 13 - lactamase / rifampicin ) STANLEY TABOR

The RNA polymerase gene of bacteriophage T7 has been cloned into the plasmid pBR322 under the inducible control of the X PL promoter. After induction, T7 RNA polymerase constitutes 20% of the soluble protein of Escherichia coli, a 200-fold increase over levels found in T7-infected cells. The overproduced enzyme has been purified to homogeneity. During extraction the enzyme is sensitive to a specific proteolysis, a reaction that can be prevented by a modification of lysis conditions. The specificity of T7 RNA polymerase for its own promoters, combined with the ability to inhibit selectively the host RNA polymerase with rifampicin, permits the exclusive expression of genes under the control of a T7 RNA polymerase promoter. We describe such a coupled system and its use to express high levels of phage T7 gene 5 protein, a subunit of T7 DNA polymerase. During bacteriophage T7 infection, the right-most 80% of the genome is transcribed by a phage-encoded RNA polymerase, the product of gene I (Fig. 1) (1). In contrast to the multisubunit RNA polymerases of bacteria and eukaryotes, T7 RNA polymerase is a single polypeptide of molecular weight 98,800 (2, 3). The enzyme is specific for its own promoters, a conserved 23-base-pair (bp) sequence (4-6). T7 RNA polymerase is present in relatively low amounts in T7-infected cells, constituting 0.1% of the cellular protein. To facilitate studies on its role in the initiation of T7 DNA replication (7), we have placed its gene on a plasmid under the control of the A PL promoter. When induced, T7 RNA polymerase constitutes 20% of the soluble protein, permitting a simple purification of the enzyme to homogeneity. Davanloo et al. (8) have also described the purification of T7 RNA polymerase from cells overexpressing the cloned T7 gene 1. A logical extension of these studies is to exploit the specificity of T7 RNA polymerase for its promoters to express other cloned genes. Transcription by Escherichia coli RNA polymerase can be inhibited selectively by the addition of rifampicin. Here, we use the T7 RNA polymerase/promoter system to overproduce bacteriophage T7 gene 5 protein, a subunit of the T7 DNA polymerase (Fig. 1). MATERIALS AND METHODS Strains. E. coli HMS262 (thrleu lacY thisupE hsdR tonAtrxA-) is E. coli C600 (9) transduced with hsdR and trxA-. E. coli B/7004 (10) was the donor of trxA-. E. coli HMS273 [lacar trpam phoar malam SupCCS rpsL tsx::TnJO lon(A)100 htpRam] is SG935 from S. Goff (Harvard Medical School). pACYC177 and pACYC184 have been described (11). pJL23, provided by J. Lodge and T. Roberts (Harvard Medical School), is a derivative of pACYC184 that contains 1 2 5 A0 .... =-iI : M -iu -