A mutant T7 phage promoter is specifically transcribed by T7‐RNA polymerase in mammalian cells

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Abstract
The phage T7 promoter/polymerase system is highly specific in bacteria in contrast to that observed in mammalian cells. A number of cell lines exhibit a considerable level of expression from the T7 promoter, even in the absence of T7‐RNA polymerase. Here, we demonstrate that nuclear‐factor‐including components of the TFIID fraction, bind to the T7 promoter and inhibit transcription by T7‐RNA polymerase. In order to increase the specificity of the promoter for T7‐RNA polymerase and to abolish binding of nuclear factors, a novel strategy for the selection of randomly mutated promoters was established. The strategy involves adsorption of mutant promoters to HeLa extracts and binding of the free oligonucleotides to T7‐RNA polymerase, cloning, and functional testing of the recombinants. After selection, the resulting mutant promoters showed an increase in specificity for transcription by T7‐RNA polymerase.