Johnson A, Meyer B J, Ptashne M
Proc Natl Acad Sci U S A. 1978 Apr;75(4):1783-7. doi: 10.1073/pnas.75.4.1783.
The mechanism of action of cro protein was probed by measuring its ability to protect DNA against methylation by dimethyl sulfate and its effect on transcription in vitro. The cro protein binds to the same three sites in the right operator (OR) of bacteriophage lambda DNA as does the lambda repressor. Dimethyl sulfate protection experiments reveal major groove contacts for both proteins, and cro protein protects from methylation a subset of those purines protected by lambda repressor. These experiments also show that the relative affinity of these two proteins for the three operator sites is different: whereas lambda repressor binds with an affinity OR1 greater than OR2 greater than OR3, the order for cro protein is OR3 greater than (OR1, OR2). As predicted by these results, cro protein, like the lambda repressor, blocks in vitro transcription of cI and cro from the two divergent promoters that overlap OR. Also as predicted, transcription of cI is turned off at lower cro protein concentrations than is transcription of cro, whereas the opposite order of repression is obtained with lambda repressor. These results describe the molecular mechanism of cro protein action and show that two regulatory proteins can bind to the same three adjacent sites in DNA with markedly different consequences.
通过测量Cro蛋白保护DNA免受硫酸二甲酯甲基化的能力及其对体外转录的影响，对其作用机制进行了探究。Cro蛋白与噬菌体λDNA右操纵子（OR）中的相同三个位点结合，就像λ阻遏物一样。硫酸二甲酯保护实验揭示了这两种蛋白质与大沟的接触，并且Cro蛋白保护的嘌呤子集是λ阻遏物所保护的那些嘌呤的一部分。这些实验还表明，这两种蛋白质对三个操纵子位点的相对亲和力不同：λ阻遏物与OR1的亲和力大于OR2大于OR3，而Cro蛋白的顺序是OR3大于（OR1，OR2）。正如这些结果所预测的，Cro蛋白与λ阻遏物一样，从与OR重叠的两个不同启动子阻断cI和cro的体外转录。同样如预测的那样，在比cro转录更低的Cro蛋白浓度下，cI转录被关闭，而对于λ阻遏物则获得相反的抑制顺序。这些结果描述了Cro蛋白作用的分子机制，并表明两种调节蛋白可以与DNA中相同的三个相邻位点结合，产生明显不同的结果。
