Lieber A, Sandig V, Strauss M
Max Planck Gesellschaft, Humboldt Universität, Max Delbrück Centrum für Molekulare Medizin, Berlin-Buch, Germany.
Eur J Biochem. 1993 Oct 1;217(1):387-94. doi: 10.1111/j.1432-1033.1993.tb18257.x.
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.
与在哺乳动物细胞中观察到的情况相比，噬菌体T7启动子/聚合酶系统在细菌中具有高度特异性。许多细胞系即使在没有T7-RNA聚合酶的情况下，也能从T7启动子高水平表达。在这里，我们证明包括TFIID组分中的核因子在内的成分，会与T7启动子结合并抑制T7-RNA聚合酶的转录。为了提高启动子对T7-RNA聚合酶的特异性并消除核因子的结合，建立了一种选择随机突变启动子的新策略。该策略包括将突变启动子吸附到HeLa提取物上，使游离寡核苷酸与T7-RNA聚合酶结合，克隆重组体并进行功能测试。经过筛选，所得突变启动子对T7-RNA聚合酶转录的特异性有所提高。
