Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes.
A gene expression system based on bacteriophage T7 RNA polymerase has been developed. T7 RNA polymerase is highly selective for its own promoters, which do not occur naturally in Escherichia coli. A relatively small amount of T7 RNA polymerase provided from a cloned copy of T7 gene 1 is sufficient to direct high-level transcription from a T7 promoter in a multicopy plasmid. Such transcription can proceed several times around the plasmid without terminating, and can be so active that transcription by E. coli RNA polymerase is greatly decreased. When a cleavage site for RNase III is introduced, discrete RNAs of plasmid length can accumulate. The natural transcription terminator from T7 DNA also works effectively in the plasmid. Both the rate of synthesis and the accumulation of RNA directed by T7 RNA polymerase can reach levels comparable with those for ribosomal RNAs in a normal cell. These high levels of accumulation suggest that the RNAs are relatively stable, perhaps in part because their great length and/or stem-and-loop structures at their 3' ends help to protect them against exonucleolytic degradation. It seems likely that a specific mRNA produced by T7 RNA polymerase can rapidly saturate the translational machinery of E. coli, so that the rate of protein synthesis from such an mRNA will depend primarily on the efficiency of its translation. When the mRNA is efficiently translated, a target protein can accumulate to greater than 50% of the total cell protein in three hours or less. We have used two ways to deliver active T7 RNA polymerase to the cell; infection by a lambda derivative that carries gene 1, or induction of a chromosomal copy of gene 1 under control of the lacUV5 promoter. When gene 1 is delivered by infection, very toxic target genes can be maintained silent in the cell until T7 RNA polymerase is introduced, when they rapidly become expressed at high levels. When gene 1 is resident in the chromosome, even the very low basal levels of T7 RNA polymerase present in the uninduced cell can prevent the establishment of plasmids carrying toxic target genes, or make the plasmid unstable.(ABSTRACT TRUNCATED AT 400 WORDS)
D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000096:biosynthesis; D013604:T-Phages / Q000201:enzymology / Q000235:genetics*; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000096:biosynthesis
D012367:RNA, Viral; D014764:Viral Proteins; D012321:DNA-Directed RNA Polymerases