Direct stimulus perception and transcription activation by a membrane-bound DNA binding protein.
Direct stimulus perception and transcription activation by a membrane-bound DNA binding protein.
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DOI：
10.1111/j.1365-2958.2009.06787.x
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发表时间：
2009-08
影响因子：
3.6
通讯作者：
Cook GM
中科院分区：
文献类型：
--
作者：
Gebhard S;Gaballa A;Helmann JD;Cook GM
很少有膜蛋白在转录调控中的作用已被研究，没有一个能够感知各自的刺激和激活转录的调节子没有辅助蛋白的帮助。粪肠球菌的杆菌肽耐药调节因子BcrR是一种膜结合DNA结合蛋白，是杆菌肽耐药基因bcrABD的杆菌肽依赖性表达所需的。在这里，我们发现BcrR直接与Zn 2 +-杆菌肽（Kd = 2-5 μM）相互作用，但不与无金属杆菌肽相互作用。基于溶液的DNA结合试验表明，BcrR对其靶DNA的亲和力比以前发现的跨膜调节剂高得多（Kd = 40 nM），与可溶性DNA结合蛋白相当。缺乏跨膜结构域的BcrR的构建体不能与DNA结合，表明膜定位对于DNA结合是重要的。杆菌肽没有引起BcrA启动子上BcrR的DNA酶I足迹的变化，但用BcrR-脂蛋白体进行的体外转录测定显示了杆菌肽依赖的转录激活。这些发现表明，BcrR是一个真正的单组分跨膜信号转导系统，它感知细胞外刺激（杆菌肽的存在），并将其中继到细胞内的转录反应，独立于任何辅助蛋白。
Few membrane proteins with a role in transcriptional regulation have been studied, and none are able to perceive their respective stimuli and activate transcription of their regulons without the aid of auxiliary proteins. The bacitracin resistance regulator, BcrR, of Enterococcus faecalis is a membrane-bound DNA-binding protein and is required for bacitracin-dependent expression of the bacitracin resistance genes, bcrABD. Here, we show that BcrR interacts directly with Zn2+-bacitracin (Kd = 2–5 μM), but not metal-free bacitracin. A solution-based DNA binding assay demonstrated that the affinity of BcrR for its target DNA is much higher (Kd = 40 nM) than previously found for transmembrane regulators and is comparable to that of soluble DNA binding proteins. A construct of BcrR that lacked the transmembrane domain was unable to bind to DNA, indicating that membrane-localization was important for DNA binding. Bacitracin did not cause a change in the DNaseI footprint of BcrR on the bcrA promoter, but in vitro transcription assays with BcrR-proteoliposomes showed bacitracin-dependent activation of transcription. These findings demonstrate that BcrR is a bona fide one-component transmembrane signal transduction system, which perceives an extracellular stimulus (presence of bacitracin) and relays it to an intracellular transcriptional response independent of any auxiliary proteins.
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影响因子：
3.6
作者：
Gaballa, Ahmed;Helmann, John D.
通讯作者：
Helmann, John D.
影响因子：
2.1
作者：
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通讯作者：
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影响因子：
3.6
作者：
Sola-Landa, A;Rodríguez-García, A;Martín, JF
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影响因子：
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作者：
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Barton, GJ
影响因子：
4.9
作者：
Manson, JM;Keis, S;Cook, GM
通讯作者：
Cook, GM