DNA-DEPENDENT RENATURATION OF AN INSOLUBLE DNA-BINDING PROTEIN - IDENTIFICATION OF THE RHAS BINDING-SITE AT RHABAD
DNA-DEPENDENT RENATURATION OF AN INSOLUBLE DNA-BINDING PROTEIN - IDENTIFICATION OF THE RHAS BINDING-SITE AT RHABAD
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DOI：
10.1006/jmbi.1994.1684
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发表时间：
1994-11-11
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
先前的工作已经表明，RhaS蛋白直接激活L-鼠李糖分解代谢操纵子rhaBAD，并且可能的RhaS结合位点位于相对于rhaBBD转录起始点的位置-84的下游。由于过度产生的RhaS蛋白的极端不溶性，RhaS结合到该DNA位点的生物化学分析是不可能的。在这里，我们已经能够直接分析的DNA结合特性的RhaS通过开发一种方法，将不溶性RhaS蛋白折叠成具有特异性的DNA结合活性的形式。我们发现，只有在DNA存在下进行复性反应，才能回收活性RhaS蛋白。我们还发现，从相关的AraC蛋白的DNA结合活性的恢复，在尿素中变性后，依赖于添加的DNA。为了测试回收的RhaS DNA结合活性的特异性，并确定结合位点以与其他AraC家族结合位点进行比较，我们随后研究了RhaS结合位点的细节。在DNA酶足迹分析中使用重折叠的RhaS蛋白，我们发现RhaS保护rhaBAD启动子的位置-83至-28的区域。对rhaBAD启动子区域中单碱基突变的影响的分析表明，RhaS结合到两个17 bp半位点的反向重复序列上，两个半位点之间相隔16 bp，位于相对于rhaBAD转录起始位点的-81和-32之间。
Previous work has indicated that the RhaS protein directly activates the L-rhamnose catabolic operon, rhaBAD, and that the likely RhaS binding site lies downstream of position -84 relative to the rhaBBD transcription start point. Biochemical analysis of RhaS binding to this DNA site had not been possible due to the extreme insolubility of overproduced RhaS protein. Here we have been able to analyze directly the DNA binding properties of RhaS by developing a method to refold insoluble RhaS protein into a form with specific DNA binding activity. We found that active RhaS protein could be recovered only if the renaturation reaction was performed in the presence of DNA. We also found that the recovery of DNA-binding activity from the related AraC protein, after denaturation in urea, was dependent upon added DNA. To test the specificity of the recovered RhaS DNA binding activity, and to define the binding site for comparison with other AraC family binding sites, ive then investigated the details of the RhaS binding site. Using refolded RhaS protein in a DNase footprinting assay, we found that RhaS protects a region of the rhaBAD promoter from position -83 to -28. Analysis of the effects of single base mutations in the rhaBAD promoter region indicates that RhaS binds to an inverted repeat of two 17 bp half-sites separated by 16 bp, located between -81 and -32 relative to the rhaBAD transcription start site.