A mutant T7 phage promoter is specifically transcribed by T7-RNA polymerase in mammalian cells.
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.
Lieber
A
A
Max Planck Gesellschaft, Humboldt Universität, Max Delbrück Centrum für Molekulare Medizin, Berlin-Buch, Germany.
Sandig
V
V
Strauss
M
M
D000818:Animals; D017123:Bacteriophage T7 / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D016466:CHO Cells / Q000378:metabolism; D006224:Cricetinae; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic; D014162:Transfection; D014764:Viral Proteins
D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases