Identification of UreR binding sites in the Enterobacteriaceae plasmid-encoded and Proteus mirabilis urease gene operons.
Identification of UreR binding sites in the Enterobacteriaceae plasmid-encoded and Proteus mirabilis urease gene operons.
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肠杆菌科质粒编码和奇异变形杆菌脲酶基因操纵子中 UreR 结合位点的鉴定。
DOI：
10.1046/j.1365-2958.1999.01283.x
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发表时间：
1999
影响因子：
3.6
通讯作者：
Collins,CM
中科院分区：
文献类型：
--
作者：
Thomas,VJ;Collins,CM
密切相关的奇异变形杆菌和肠杆菌质粒编码的尿素酶基因受到AraC样转录激活因子UreR的正调控。在效应分子尿素的存在下，UreR促进尿素酶操纵子中的起始基因ofureD的转录，并增加趋异转录的dureR的转录。在这里，我们确定了ureRp-ureDp基因间区域的UreR特异性结合位点。表达并纯化重组UreR（rUreR），并用该蛋白进行凝胶迁移和DNA酶I保护试验。这些分析表明，在质粒编码的和P.紫茉莉ureRp-ureDp基因间区。从DNA酶I保护试验预测TA/GT/CA/TT/GC/TTA/TT/AATTG的共有结合位点。尽管rUreR在尿素存在和不存在下都与特异性DNA结合位点结合，但当尿素存在时，rUreR-DNA复合物相互作用的解离速率常数k-1可测量地不同。在不存在尿素的情况下，蛋白质-DNA复合物的解离，对于两个Rp和dureDp，在最早的时间点完成，并且不可能确定速率。在存在尿素的情况下，rUreR-ureRp相互作用的解离可通过ak-1测量，其为1.2 ± 0.2 × 10−2s− 1，rUreR-ureDp相互作用的解离可通过ak-1测量，其为2.6 ± 0.1 × 10−3s−1。 这相当于ureRp-rUreR相互作用的半衰期为58 s，ureDp-rUreR相互作用的半衰期为4 min 26 s。 提出了一个模型，描述尿素在这些启动子的激活中的潜在作用。
The closely relatedProteus mirabilisandEnterobacteriaceaeplasmid‐encoded urease genes are positively regulated by the AraC‐like transcriptional activator UreR. In the presence of the effector molecule urea, UreR promotes transcription ofureD, the initial gene in the urease operon, and increases transcription of the divergently transcribedureR. Here, we identify UreR‐specific binding sites in theureRp–ureDp intergenic regions. Recombinant UreR (rUreR) was expressed and purified, and gel shift and DNase I protection assays were performed with this protein. These analyses indicated that there are two distinct rUreR binding sites in both the plasmid‐encoded andP. mirabilis ureRp–ureDp intergenic regions. A consensus binding site of TA/GT/CA/TT/GC/TTA/TT/AATTG was predicted from the DNase I protection assays. Although rUreR bound to the specific DNA binding site in both the presence and the absence of urea, the dissociation rate constantk–1of the rUreR–DNA complex interaction was measurably different when urea was present. In the absence of urea, the dissociation of the protein–DNA complexes, for bothureRp andureDp, was complete at the earliest time point, and it was not possible to determine a rate. In the presence of urea, dissociation was measurable with ak–1for the rUreR–ureRp interaction of 1.2 ± 0.2 × 10−2s−1and ak–1for the rUreR–ureDp interaction of 2.6 ± 0.1 × 10−3s−1. This corresponds to a half‐life of theureRp–rUreR interaction of 58 s, and a half‐life of theureDp–rUreR interaction of 4 min 26 s. A model describing a potential role for urea in the activation of these promoters is proposed.