Linking structural assembly to gene expression:: a novel mechanism for regulating the activity of a σ54 transcription factor
Linking structural assembly to gene expression:: a novel mechanism for regulating the activity of a σ54 transcription factor
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DOI：
10.1111/j.1365-2958.2005.04857.x
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发表时间：
2005-11-01
影响因子：
3.6
通讯作者：
Gober, JW
中科院分区：
文献类型：
--
作者：
Dutton, RJ;Xu, ZH;Gober, JW
在新月藻中，晚鞭毛基因的时间和空间表达受Sigma(54)转录激活因子FlbD的调节。遗传实验表明，反式作用因子Flix调节FlbD以响应鞭毛组装的进展，抑制FlbD的活性，直到早期的鞭毛基体结构组装完成。在组装这种结构后，Flix被认为是FlbD的激活剂。在这里，我们研究了Flix介导的FlbD活性的调节机制。体外转录实验表明，纯化的Flix可以抑制FlbD激活的转录。由FlbD的功能获得突变体(FlbD-1204)激活的转录，在缺乏早期鞭毛结构的情况下在体内活跃，抵抗Flix的抑制作用。DNA结合研究表明，Flix抑制了野生型FlbD与增强子DNA的相互作用，但不影响FlbD催化的ATPase活性。Flix对FlbD-1204的DNA结合活性没有影响，表明该突变蛋白通过逃避Flix介导的负调控，绕过了早期鞭毛组装的转录要求。凝胶过滤和免疫共沉淀实验表明，Flix与FlbD形成稳定的络合物。这些实验表明，在研究得很好的sigma(54)转录激活子中，FlbD活性的调节是不寻常的，显然将一个双组分的接收域与通过与伙伴蛋白Flix相互作用施加的额外控制结合在一起。
In Caulobacter crescentus, the temporal and spatial expression of late flagellar genes is regulated by the sigma(54) transcriptional activator, FlbD. Genetic experiments have indicated that the trans-acting factor FliX regulates FlbD in response to the progression of flagellar assembly, repressing FlbD activity until an early flagellar basal body structure is assembled. Following assembly of this structure, FliX is thought to function as an activator of FlbD. Here we have investigated the mechanism of FliX-mediated regulation of FlbD activity. In vitro transcription experiments showed that purified FliX could function as a repressor of FlbD-activated transcription. Transcription activated by a gain-of-function mutant of FlbD (FlbD-1204) that is active in vivo in the absence of an early flagellar structure, was resistant to the repressive effects of FliX. DNA binding studies showed that FliX inhibited the interaction of wild-type FlbD with enhancer DNA but did not effect FlbD-catalysed ATPase activity. DNA binding activity of FlbD-1204 was relatively unaffected by FliX indicating that this mutant protein bypasses the transcriptional requirement for early flagellar assembly by escaping FliX-mediated negative regulation. Gel filtration and co-immunoprecipitation experiments indicated that FliX formed a stable complex with FlbD. These experiments demonstrate that regulation of FlbD activity is unusual among the well-studied sigma(54) transcriptional activators, apparently combining a two-component receiver domain with additional control imposed via interaction with a partner protein, FliX.