Lurz R, Heisig A, Velleman M, Dobrinski B, Schuster H
Max-Planck-Institut für Molekulare Genetik, Berlin, Federal Republic of Germany.
J Biol Chem. 1987 Dec 5;262(34):16575-9.
Repression of a strong promoter localized 5' to the P1 ban gene is a prerequisite for cloning the ban operon in the multicopy plasmid pBR325. Repression is brought about by the binding of P1 repressor to the operator of the ban operon (Heisig, A., Severin, I., Seefluth, A. K., and Schuster, H. (1987) Mol. Gen. Genet. 206, 368-376). Binding of RNA polymerase in vitro overlaps with the operator and is inhibited by P1 repressor as shown by electron microscopy. The mutant P1 bac, which renders ban expression constitutive, contains a single base pair exchange within the operator. As a consequence, more repressor is required (i) for the inhibition of binding of RNA polymerase, and (ii) for the electrophoretic retardation of a P1 bac DNA fragment when compared to the corresponding bac+ fragment. A P1 ban recombinant plasmid containing a 4-base pair deletion close to the operator still allows binding of repressor but not of RNA polymerase. By that means, a repressible promoter is located at the P1 map position 72 in a distance of about 2.5 kilobase pairs to the beginning of the ban gene.
将位于P1 ban基因5'端的强启动子抑制是在多拷贝质粒pBR325中克隆ban操纵子的前提条件。这种抑制是通过P1阻遏物与ban操纵子的操纵基因结合来实现的(Heisig, A., Severin, I., Seefluth, A. K., and Schuster, H. (1987) Mol. Gen. Genet. 206, 368 - 376)。如电子显微镜所示,体外RNA聚合酶的结合与操纵基因重叠,并受到P1阻遏物的抑制。使ban表达组成型的突变体P1 bac在操纵基因内含有一个单碱基对交换。因此,与相应的bac +片段相比,(i)抑制RNA聚合酶结合以及(ii)使P1 bac DNA片段电泳阻滞需要更多的阻遏物。一个在靠近操纵基因处含有4个碱基对缺失的P1 ban重组质粒仍然允许阻遏物结合,但不允许RNA聚合酶结合。通过这种方式,一个可阻遏的启动子位于P1图谱位置72,距离ban基因起始约2.5千碱基对。