Lehming N, Sartorius J, Niemöller M, Genenger G, v Wilcken-Bergmann B, Müller-Hill B
Institut für Genetik der Universität zu Köln, FRG.
EMBO J. 1987 Oct;6(10):3145-53. doi: 10.1002/j.1460-2075.1987.tb02625.x.
We have constructed a system which allows systematic testing of repressor--operator interactions. The system consists of two plasmids. One of them carries a lac operon in which lac operator has been replaced by a unique restriction site into which synthetic operators can be cloned. The other plasmid carries the gene coding for the repressor, in our case a semisynthetic lacI gene of which parts can be exchanged in a cassette-like manner. A galE host allows us to select for mutants which express repressors with altered specificities. Here we report the change of specificity in the lac system by changing residues 1 and 2 of the recognition helix of lac repressor. The specificity changes are brought about cooperatively by the change of both residues. Exchanges of just one residue broaden the specificity. Our results hint that the recognition helix of lac repressor may possibly have the opposite orientation to those in Lambda cro protein or 434 CI repressor.
我们构建了一个能够对阻遏物 - 操纵子相互作用进行系统测试的系统。该系统由两个质粒组成。其中一个携带一个乳糖操纵子，在这个操纵子中，乳糖操纵基因已被一个独特的限制酶切位点所取代，合成的操纵基因可以克隆到这个位点中。另一个质粒携带编码阻遏物的基因，在我们的例子中是一个半合成的乳糖抑制因子基因，其部分片段可以以盒式方式进行交换。一个galE宿主使我们能够筛选出表达具有改变特异性的阻遏物的突变体。在此我们报告通过改变乳糖阻遏物识别螺旋的第1和第2位残基，乳糖系统中的特异性发生了变化。这两个残基的变化协同导致了特异性的改变。仅交换一个残基会拓宽特异性。我们的结果表明，乳糖阻遏物的识别螺旋可能与λ噬菌体cro蛋白或434 CI阻遏物中的识别螺旋方向相反。
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