A novel mechanism controls anaerobic and catabolite regulation of the Escherichia coli tdc operon
A novel mechanism controls anaerobic and catabolite regulation of the Escherichia coli tdc operon
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DOI：
10.1111/j.1365-2958.2001.02316.x
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发表时间：
2001-03
影响因子：
3.6
通讯作者：
G. Sawers
中科院分区：
文献类型：
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作者：
G. Sawers
tdc操纵子受到CRP控制的分解代谢物抑制。操纵子的表达也被厌氧诱导，尽管这种调节不依赖于FNR或ArcA的直接控制。最近，发现tdc操纵子的厌氧表达在葡萄糖存在下由从革兰氏阳性厌氧菌丁酸梭菌分离的异源基因偶然诱导。该基因被称为tcbC，编码14.5 kDa的组蛋白样蛋白。使用tdc-lacZ融合，结果表明，TcbC没有激活tdc表达功能取代任何操纵子调节。用RNA聚合酶和CRP进行的体外转录分析显示，忠实的CRP依赖性转录起始仅发生在超螺旋模板上。在松弛或线性DNA模板上未观察到特异性CRP依赖性转录起始。令人惊讶的是，纯化的His标记的TcbC从松弛的环状模板激活转录，但不是从超螺旋或线性模板。检查的CRP结合位点的TDC启动子显示，它位于转录起始位点的上游43.5 bp。将CRP位点重新定位在-41.5 bp处，消除了TcbC蛋白的激活，并允许在线性、松弛和超螺旋模板上发生CRP依赖性转录。TcbC非特异性结合DNA;然而，在拓扑异构酶I松弛试验中，证明TcbC对负超螺旋DNA施加扭转约束，这影响酶松弛拓扑异构体的能力。综上所述，这些结果强烈表明TcbC通过改变tdc启动子的局部拓扑状态激活tdc的转录，并且在野生型tdc启动子中，CRP结合位点未对齐以允许转录仅在最佳条件下发生。事实上，体内转录分析显示，CRP结合位点重新定位到− 41.5 bp导致高水平的CRP依赖性转录，即使在分解代谢物抑制条件下，转录也不再受TcbC的影响。然而，值得注意的是，突变启动子的厌氧调节被保留。这表明其他tdc调节因子TdcA和TdcR通过CRP控制厌氧转录激活。
The tdc operon is subject to CRP‐controlled catabolite repression. Expression of the operon is also induced anaerobically, although this regulation does not rely on direct control by either FNR or ArcA. Recently, the anaerobic expression of the tdc operon was found to be fortuitously induced in the presence of glucose by a heterologous gene isolated from the Gram‐positive anaerobe Clostridium butyricum. The gene, termed tcbC, encoded a histone‐like protein of 14.5 kDa. Using tdc–lacZ fusions, it was shown that TcbC did not activate tdc expression by functionally replacing any of the operon regulators. In vitro transcription analyses with RNA polymerase and CRP revealed that faithful CRP‐dependent transcription initiation occurred only on supercoiled templates. No specific, CRP‐dependent transcription initiation was observed on relaxed or linear DNA templates. Surprisingly, purified His‐tagged TcbC activated transcription from a relaxed, circular template, but not from supercoiled or linear templates. Examination of the CRP binding site of the tdc promoter revealed that it was located 43.5 bp upstream of the transcription initiation site. Repositioning of the CRP site at −41.5 bp abolished activation by the TcbC protein and allowed CRP‐dependent transcription to occur on linear, relaxed and supercoiled templates. TcbC bound DNA non‐specifically; however, in topoisomerase I relaxation assays, it was demonstrated that TcbC imposed torsional constraints on negatively supercoiled DNA, which influenced the ability of the enzyme to relax the topoisomers. Taken together, these results strongly suggest that TcbC activates transcription of tdc by altering the local topological status of the tdc promoter and that, in the wild‐type tdc promoter, the CRP binding site is misaligned to allow transcription to occur only under optimal conditions. Indeed, in vivo transcription analyses revealed that repositioning of the CRP binding site to −41.5 bp resulted in high‐level, CRP‐dependent transcription, even under catabolite‐repressing conditions, and that transcription was no longer influenced by TcbC. Remarkably, however, anaerobic regulation of the mutant promoter was retained. This indicates that the other tdc regulators, TdcA and TdcR, govern anaerobic transcription activation by CRP.