A REGULATORY CASCADE IN THE INDUCTION OF RHABAD
A REGULATORY CASCADE IN THE INDUCTION OF RHABAD
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DOI：
10.1006/jmbi.1993.1565
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发表时间：
1993-11-05
影响因子：
5.6
通讯作者：
SCHLEIF, RF
中科院分区：
文献类型：
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作者：
EGAN, SM;SCHLEIF, RF
RhaS和RhaR调节蛋白编码于大肠杆菌-鼠李糖基因簇中。我们使用互补分析和DNA迁移率变动分析来证明RhaR不是鼠李糖分解代谢操纵子rhaBAD的直接激活剂。rhaS的框内缺失（rhS-rhaR+）消除了haBAD启动子pBAD的表达，而rhaS的过表达大大加快了通常缓慢的从mpBAD转录的诱导。在转录-翻译偶联试验中，只有当加入rhaS +DNA以允许RhaS蛋白合成时才检测到来自于mpBAD的表达。因此，RhaS似乎是rhaBAD表达的直接l-鼠李糖特异性激活剂。缺失作图将1-鼠李糖特异性调节子的结合位点定位于相对于haBAD转录起始位点的-70位重叠区域。缺失图谱和DNA迁移率变化测定在鼠李糖特异性调节剂结合位点的上游定位了CRP结合位点。定量引物延伸分析表明，两个theraBAD和rhaSR信息的诱导异常缓慢，需要40至50分钟才能达到稳态水平。rhaBAD的诱导显然涉及一个调节级联反应，其中RhaR首先诱导rhaSR表达，然后RhaS积累并诱导rhaBAD表达。
The RhaS and RhaR regulatory proteins are encoded in theEscherichia colil-rhamnose gene cluster. We used complementation analysis and DNA mobility shift assays to show that RhaR is not the direct activator of thel-rhamnose catabolic operon,rhaBAD. An inframe deletion ofrhaS(rhS-rhaR+) eliminated expression from therhaBADpromoter,pBAD, while overexpression ofrhaSgreatly speeded the normally slow induction of transcription frompBAD. Expression frompBADin a coupled transcription-translation assay was only detected whenrhaS+DNA was added to allow synthesis of RhaS protein. RhaS thus appears to be the directl-rhamnose-specific activator ofrhaBADexpression. Deletion mapping located the binding site for thel-rhamnose-specific regulator to a region overlapping position -70 relative to therhaBADtranscription start site. Deletion mapping and DNA mobility shift assays located a CRP binding site just upstream from the binding site for thel-rhamnose specific regulator. Quantitative primer extension analysis showed that induction of both therhaBADandrhaSRmessages was unusually slow, requiring 40 to 50 minutes to reach a steady state level. Induction ofrhaBADapparently involves a regulatory cascade in which RhaR first inducesrhaSRexpression, then RhaS accumulates and inducesrhaBADexpression.