Fujita Y, Miwa Y
Department of Biotechnology, Faculty of Engineering, Fukuyama University, Japan.
J Biol Chem. 1989 Mar 5;264(7):4201-6.
Expression of the Bacillus subtilis gluconate (gnt) operon is negatively regulated by the gnt repressor which is antagonized by D-gluconate or D-glucono-delta-lactone. The repressor specifically binds to a gnt operator near the gnt promoter. From the results of gel retardation assaying of the gnt repressor and various restriction enzyme digests of a fragment carrying the gnt promoter and the gntR gene, the gnt operator was found to be located within a 141-base pair region containing the gnt promoter. Deoxyribonuclease I protection experiments revealed that the gnt repressor protected a gnt promoter region, between -10 and +15, which contains a region showing dyad symmetry with a sequence showing half-symmetry, ATACTTGTA. A 35-base pair synthetic duplex DNA containing this region showing dyad symmetry specifically bound to the gnt repressor. Through cleavage at two AccI sites in the protected region an 8-base pair deletion was introduced into the region showing dyad symmetry, which made expression of the gnt promoter constitutive. A DNA fragment carrying this deletion did not bind to the repressor. These results clearly indicated that the gnt operator sequence is a sequence containing the region showing dyad symmetry located at the transcription initiation site of the gnt operon. It was also suggested that the gnt operon contains only one operator. The similarity in the recognition between the repressor-operator interaction of the gnt operon and those of Escherichia coli TrpR and its operators was discussed.
枯草芽孢杆菌葡萄糖酸盐（gnt）操纵子的表达受gnt阻遏物负调控，D - 葡萄糖酸盐或D - 葡萄糖酸 - δ - 内酯可拮抗该阻遏物。该阻遏物特异性结合gnt启动子附近的gnt操纵基因。通过对gnt阻遏物进行凝胶阻滞分析以及对携带gnt启动子和gntR基因的片段进行各种限制性酶切，发现gnt操纵基因位于一个包含gnt启动子的141碱基对区域内。脱氧核糖核酸酶I保护实验表明，gnt阻遏物保护了gnt启动子区域，即 - 10至 + 15之间，该区域包含一个具有二重对称的区域以及一个半对称序列ATACTTGTA。一个包含该二重对称区域的35碱基对合成双链DNA特异性结合gnt阻遏物。通过在受保护区域的两个AccI位点进行切割，在具有二重对称的区域引入了一个8碱基对的缺失，这使得gnt启动子的表达组成型。携带该缺失的DNA片段不与阻遏物结合。这些结果清楚地表明，gnt操纵基因序列是一个包含位于gnt操纵子转录起始位点的二重对称区域的序列。还表明gnt操纵子仅包含一个操纵基因。讨论了gnt操纵子的阻遏物 - 操纵基因相互作用与大肠杆菌TrpR及其操纵基因之间识别的相似性。
