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 lin es exhibit a considerable level of expression from the T7 promoter, ev en in the absence of T7-RNA polymerase. Here, we demonstrate that nucl ear-factor-including components of the TFIID fraction, bind to the T7 promoter and inhibit transcription by T7-RNA polymerase. In order to i ncrease the specificity of the promoter for T7-RNA polymerase and to a bolish binding of nuclear factors, a novel strategy for the selection of randomly mutated promoters was established. The strategy involves a dsorption of mutant promoters to HeLa extracts and binding of the free oligonucleotides to T7-RNA polymerase, cloning, and functional testin g of the recombinants. After selection, the resulting mutant promoters showed an increase in specificity for transcription by T7-RNA polymer ase.