Creation of a T7 autogene. Cloning and expression of the gene for bacteriophage T7 RNA polymerase under control of its cognate promoter.
The coding sequence for bacteriophage T7 RNA polymerase has been cloned and expressed under control of a cognate T7 promoter, a configuration referred to as an autogene. Cloning a T7 autogene in a derivative of plasmid pBR322 in Escherichia coli was achieved by a combination of blocking initiation at the T7 promoter with bound lac repressor and inhibiting the polymerase itself by T7 lysozyme. Neither type of inhibition by itself was sufficient to control the autogene. Upon unblocking the T7 promoter with added inducer. T7 RNA polymerase produced its own mRNA, leading to autocatalytic production of polymerase protein. T7 autogenes may be useful for developing high-level gene expression systems in a variety of cell types, with little if any need for the host cell RNA polymerase.
Dubendorff
J W
JW
Biology Department, Brookhaven National Laboratory, Upton, NY 11973.
Studier
F W
FW
D003001:Cloning, Molecular / Q000379:methods; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis / Q000235:genetics*; D004790:Enzyme Induction; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral / Q000187:drug effects; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D012293:Rifampin / Q000494:pharmacology; D013604:T-Phages / Q000201:enzymology / Q000235:genetics*
D012321:DNA-Directed RNA Polymerases; D012293:Rifampin