Shirabe K, Ebina Y, Miki T, Nakazawa T, Nakazawa A
Nucleic Acids Res. 1985 Jul 11;13(13):4687-98. doi: 10.1093/nar/13.13.4687.
In previous experiments, we showed that the in vivo transcription of the colicin E1 gene was dependent on cyclic AMP in adenylate cyclase-defective mutant cells of Escherichia coli (Ebina, Y. and Nakazawa, A (1983) J. Biol. Chem. 258, 7072-7078). We now show that cyclic AMP and cyclic AMP receptor protein stimulated the in vitro transcription of the gene in the presence of spermidine. As determined in DNase I protection experiments, two binding sites for the complex of cyclic AMP and the receptor protein were identified about 60 base pairs (CRP-1) and 110 base pairs (CRP-2) upstream from the transcription initiation site of the colicin E1 gene. CRP-1 had a higher affinity for the complex than that of CRP-2. Substituting an unrelated DNA sequence for CRP-2 reduced the efficiency of in vitro stimulation of the gene by cyclic AMP and the receptor protein. These potential binding sites for the cyclic AMP-cyclic AMP receptor protein complex probably participate in the stimulation of the colicin E1 gene transcription.
在先前的实验中，我们发现大肠杆菌腺苷酸环化酶缺陷型突变细胞中，大肠杆菌素E1基因的体内转录依赖于环腺苷酸（江名洋和中泽明，1983年，《生物化学杂志》258卷，7072 - 7078页）。现在我们发现，在亚精胺存在的情况下，环腺苷酸和环腺苷酸受体蛋白能刺激该基因的体外转录。通过DNA酶I保护实验确定，在大肠杆菌素E1基因转录起始位点上游约60个碱基对（CRP - 1）和110个碱基对（CRP - 2）处，鉴定出了环腺苷酸与受体蛋白复合物的两个结合位点。CRP - 1对该复合物的亲和力高于CRP - 2。用无关DNA序列替换CRP - 2会降低环腺苷酸和受体蛋白对该基因体外刺激的效率。环腺苷酸 - 环腺苷酸受体蛋白复合物的这些潜在结合位点可能参与了对大肠杆菌素E1基因转录的刺激。
