碳代谢总体调控蛋白对氮代谢glnAp2启动子的调控机理

In Escherichia coli, glnA (encoding glutamine synthetase) is transcribed from two promoters (glnAp1 and glnAp2). The glnAp1 is a s70-dependent promoter that is activated by the cAMP receptor protein (CRP). Under nitrogen-deficient growth conditions, glnAp1 is repressed by NtrC-phosphate. The downstream glnAp2 promoter is s54-dependent and is activated by NtrC-phosphate. Here, we show that glnAp2 expression is affected by different carbon sources and that the CRP-cAMP complex inhibits the glnAp2 promoter activity. Primer extension and KMnO4 footprinting analysis indicate that the inhibitory effect is at the transcriptional level in vivo. When glnAp2 is activated by NifA, a similar inhibitory effect by CRP-cAMP is observed. Site-directed mutagenesis and deletion analysis indicate that the characterized and putative CRP-binding sites located in the upstream region of the glnAp2 promoter are not essential for the inhibitory effect. CRP-cAMP inhibits s54-dependent glnAp2 strongly, by 21-fold. By activating glnAp1 and downregulating glnAp2, the overall effect of CRP-cAMP on glnA expression is an approximately fourfold reduction, which correlates with the reduction of g-glutamyl transferase activities in the cells. We propose therefore that a physiological role of CRP-cAMP activation of glnAp1 is to partially compensate for CRP-cAMP downregulation of glnAp2, allowing a low but non-negligible level of expression of the important genes transcribed from it. A novel regulatory linkage between carbon and nitrogen regulons is proposed.

申请人在过去几年的研究工作中发现在大肠杆菌内环腺苷酸受体蛋白对依赖σ54RNA聚合酶膁ctA操纵子活性有抑制作用。本项目以此出发点，以大肠杆菌同源glnALG操纵子为模型，臃肿铀缴涎芯炕废佘账崾芴宓鞍锥詆lnALG操纵子活性的抑制作用的机理，试图提示环腺账崾芴宓鞍资橇堤肌⒌坏骺氐囊桓鲋匾骺匾蜃印