In vitro selection of optimal AbrB-binding sites: Comparison to known in vivo sites indicates flexibility in AbrB binding and recognition of three-dimensional DNA structures
In vitro selection of optimal AbrB-binding sites: Comparison to known in vivo sites indicates flexibility in AbrB binding and recognition of three-dimensional DNA structures
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DOI：
10.1046/j.1365-2958.1996.358882.x
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发表时间：
1996-01-01
影响因子：
3.6
通讯作者：
Strauch, MA
中科院分区：
文献类型：
--
作者：
Xu, K;Strauch, MA
枯草芽孢杆菌的AbrB蛋白主要通过与含有转录启动子的DNA区域的特异性结合相互作用来调节许多基因的表达。虽然超过15个目标区域的AbrB结合染色体定位的序列已被分析的DNA酶I足迹，没有明显的共识序列或基序尚未出现从他们的检查。使用体外选择技术，我们已经分离出最佳的AbrB-结合位点的寡核苷酸含有22或44个随机碱基对。最好的这些网站有一个明显的体外Kd，这是一个类似的大小的DNA片段，含有相应的Spo 0 E基因上的AbrB结合位点的序列低五倍。我们在体内测试了其中一个位点，发现它赋予了一个通常不受AbrB调控的启动子AbrB介导的控制。在四个独立的试验中，所选择的位点具有收敛到简单共识的基序。有人认为，这些图案的性质和间距产生一种类型的三维DNA结构，可识别的AbrB，和已知的在体内的网站，缺乏这些图案，具有近似的最佳结构的决定因素。
The AbrB protein of Bacillus subtilis regulates expression of numerous genes, primarily through specific binding interactions to DNA regions containing transcriptional promoters. Although over 15 target regions for AbrB binding to chromosomally located sequences have been analysed by DNase I footprinting, no obvious consensus sequence or motif has yet emerged from their examination. Using in vitro selection techniques, we have isolated optimal AbrB-binding sites from oligonucleotides containing 22 or 44 random base pairs. The best of these sites have an apparent in vitro K-d which is fivefold lower than a similar-sized DNA fragment containing the sequence corresponding to the AbrB-binding site on the spo0E gene. We tested one of the sites in vivo and found that it confers AbrB-mediated control upon a promoter not normally regulated by AbrB. In each of four separate trials, the selected sites possess motifs that converge to a simple consensus. It is argued that the nature and spacing of these motifs produce a type of three-dimensional DNA structure recognizable by AbrB, and that known in vivo sites, which lack these motifs, possess an approximation of the optimal structural determinant.