Positive and negative transcriptional regulation of the Escherichia coli gluconate regulon gene gntT by GntR and the cyclic AMP (cAMP)-cAMP receptor protein complex
Positive and negative transcriptional regulation of the Escherichia coli gluconate regulon gene gntT by GntR and the cyclic AMP (cAMP)-cAMP receptor protein complex
复制标题
DOI：
10.1128/jb.180.7.1777-1785.1998
复制
发表时间：
1998-04-01
影响因子：
3.2
通讯作者：
Conway, T
中科院分区：
文献类型：
--
作者：
Peekhaus, N;Conway, T
大肠杆菌的gntT基因被葡萄糖酸盐特异性诱导，并通过分解代谢物阻遏而被阻遏。因此，葡萄糖酸盐既是gntT表达的诱导物又是gntT表达的阻遏物，因为葡萄糖酸盐是抑制分解产物的糖。在gntR缺失突变体中，染色体gntT：：lacZ融合的表达是高的和组成性的，证实了GntR是gntT的负调节因子。事实上，GntR结合到两个共有的gnt操纵基因位点;一个与gntT启动子的-10区域重叠，另一个相对于转录起始位点集中在+120。GntR与这些位点的结合在体外通过凝胶还原试验和在体内通过结合位点的定点诱变来证明。GntR与操作者的结合被葡萄糖酸盐和浓度高10倍的6-磷酸葡萄糖酸盐消除。有趣的是，当gntR缺失菌株在葡萄糖酸盐存在下生长时，gntT表达降低两倍，这与分解代谢物阻遏和GntR与操纵基因位点的结合无关。gntR突变体对诱导剂的这种新反应称为超阻遏。通过环AMP（cAMP）-cAMP受体蛋白（CRP）复合物与位于gntT转录起始位点上游-71的CRP结合位点结合来激活gntT的转录。
The gntT gene of Escherichia coli Is specifically induced by gluconate and repressed via catabolite repression. Thus, gluconate is both an inducer and a repressor of gntT expression since gluconate is a catabolite-repressing sugar. In a gntR deletion mutant, the expression of a chromosomal gntT::lacZ fusion is both high and constitutive, confirming that GntR is the negative regulator of gntT. Indeed, GntR binds to two consensus gnt operator sites; one overlaps the -10 region of the gntT promoter, and the other is centered at +120 with respect to the transcriptional start site. The binding of GntR to these sites was proven in vitro by gel redardation assays and in vivo by site-directed mutagenesis of the binding sites. Binding of GntR to the operators is eliminated by gluconate and also by 6-phosphogluconate at a 10-fold-higher concentration. Interestingly, when gntR deletion strains are grown in the presence of gluconate, there is a twofold decrease in gntT expression which is independent of catabolite repression and binding of GntR to the operator sites. This novel response of gntR mutants to the inducer is termed ultrarepression. Transcription of gntT is activated by binding of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex to a CRP binding site positioned at -71 upstream of the gntT transcription start site.