A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes
(T7 DNA polymerase/T7 gene 5 protein/proteolysis/13-lactamase/rifampicin)
STANLEY TABOR AND CHARLES C. RICHARDSON
Department of Biological Chemistry, Harvard Medical School, Boston, MA 02115
Contributedby Charles C. Richardson, October 22, 1984
ABSTRACT 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 tohomogeneity. 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 describesuch a coupled system and its use to express high levels of phage T7 gene 5 protein, a subunit of T7 DNA polymerase.
-iI : =
A0 M -iu
m T7 RNA Polymerose
+ 1.1 A
-7 DNA Polymerose
S u b uni t
16871 228 7Q 22928
During bacteriophage T7 infection, the right-most 80% of thegenome 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 inT7-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 thepurification 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 bacteriophageT7 gene 5 protein, a subunit of the T7 DNA polymerase (Fig. 1).
FIG. 1. The genetic map of bacteriophage T7 with inserts showing the cloned DNA fragments used in this study. T7 base pairs are numbered as described (6). Gene sizes reflect molecular weights of protein products. 01.1A, 41.1B, and 410 correspond to the T7 RNA polymerase promoters located before genes 1.1 and 10, respectively.MATERIALS AND METHODS
Strains. E. coli HMS262 (thr- leu lac Y thi- supE hsdR tonA- trxA-) 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
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the X repressor gene c1857. HMS273/pJL23 is E. coli HMS273 containing the plasmid pJL23. pKB280-cI857, a...