Tagami H, Inada T, Kunimura T, Aiba H
Department of Molecular Biology, School of Science, Nagoya University, Japan.
Mol Microbiol. 1995 Jul;17(2):251-8. doi: 10.1111/j.1365-2958.1995.mmi_17020251.x.
CRP-cAMP-dependent operons of Escherichia coli can be expressed in cells lacking functional adenylate cyclase when they carry a second-site mutation in the crp gene (crp*). It is known that the expression of these operons is repressed by glucose, but the molecular mechanism underlying this cAMP-independent catabolite repression has been a long-standing mystery. Here we address the question of how glucose inhibits the expression of beta-galactosidase in the absence of cAMP. We have isolated several mutations in the crp gene that confer a CRP* phenotype. The expression of beta-galactosidase is reduced by glucose in cells carrying these mutations. Using Western blotting and/or SDS-PAGE analysis, we demonstrate that glucose lowers the cellular concentration of CRP* through a reduction in crp* mRNA levels. The level of CRP* protein correlates with beta-galactosidase activity. When the crp promoter is replaced with the bla promoter, the inhibitory effect of glucose on crp* expression is virtually abolished. These data strongly suggest that the lowered level of CRP* caused by glucose mediates catabolite repression in cya- crp* cells and that the autoregulatory circuit of the crp gene is involved in the down-regulation of CRP* expression by glucose.
大肠杆菌中依赖CRP-cAMP的操纵子在缺乏功能性腺苷酸环化酶的细胞中携带crp基因的第二位点突变（crp*）时可以表达。已知这些操纵子的表达受到葡萄糖的抑制，但这种不依赖cAMP的分解代谢物阻遏的分子机制一直是个长期存在的谜团。在这里，我们探讨了在没有cAMP的情况下葡萄糖如何抑制β-半乳糖苷酶表达的问题。我们分离出了crp基因中的几个赋予CRP表型的突变。在携带这些突变的细胞中，葡萄糖会降低β-半乳糖苷酶的表达。通过蛋白质印迹法和/或SDS-PAGE分析，我们证明葡萄糖通过降低crp mRNA水平来降低细胞中CRP的浓度。CRP蛋白水平与β-半乳糖苷酶活性相关。当crp启动子被bla启动子取代时，葡萄糖对crp表达的抑制作用几乎消除。这些数据强烈表明，葡萄糖导致的CRP水平降低介导了cya-crp细胞中的分解代谢物阻遏，并且crp基因的自动调节回路参与了葡萄糖对CRP表达的下调。
