In vitro reconstitution of osmoregulated expression of proU of Escherichia coli.
In vitro reconstitution of osmoregulated expression of proU of Escherichia coli.
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大肠杆菌 proU 渗透调节表达的体外重建。
DOI：
10.1073/pnas.86.4.1153
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发表时间：
1989
影响因子：
11.1
通讯作者：
Villarejo,M
中科院分区：
文献类型：
--
作者：
Ramirez,RM;Prince,WS;Bremer,E;Villarejo,M
渗透调节的proU表达已在无细胞系统中重建。proU在大肠杆菌中编码一种可诱导的、高亲和性的转运系统，用于保护甜菜碱。以前，proU-lacZ融合基因已被克隆，产生质粒pOS 3。低拷贝数的这种染色体外proU-lacZ融合基因的体内转录调控表明，质粒编码的融合体含有在渗透胁迫诱导和甘氨酸甜菜碱阻遏期间正确接收转录调控信号的顺式所有必需序列。使用无细胞（S-30）提取物，然后使用质粒pOS 3在体外编程蛋白质合成。离子化合物谷氨酸钾以浓度依赖性方式特异性刺激proU-lacZ表达。醋酸钾也诱导一些proU表达，但其他盐无效，从而排除了离子强度作为刺激信号。高浓度的蔗糖，海藻糖，或甘氨酸甜菜碱也没有诱导proU在体外表达，消除渗透压本身作为刺激。在无细胞系统中的重建排除了依赖于膨压、跨膜信号传导或在渗透性上激后合成的反式作用调节剂的膜调节机制。
Osmoregulated expression of proU has been reconstituted in a cell-free system. proU encodes an osmotically inducible, high-affinity transport system for the osmoprotectant glycine betaine in Escherichia coli. Previously, a proU-lacZ fusion gene had been cloned, resulting in plasmid pOS3. In vivo osmoregulation of this extrachromosomal proU-lacZ fusion gene at low copy number showed that the plasmid-encoded fusion contained all the necessary sequences in cis for correctly receiving osmoregulatory signals during induction by osmotic stress and repression by glycine betaine. Using a cell-free (S-30) extract, plasmid pOS3 was then used to program protein synthesis in vitro. The ionic compound potassium glutamate specifically stimulated proU-lacZ expression in a concentration-dependent manner. Potassium acetate also induced some proU expression, but other salts were ineffective, thereby ruling out ionic strength as the stimulatory signal. High concentrations of sucrose, trehalose, or glycine betaine did not induce proU expression in vitro either, eliminating osmolarity per se as the stimulus. Reconstitution in a cell-free system rules out osmoregulatory mechanisms that depend on turgor, trans-membrane signaling, or trans-acting regulators synthesized after osmotic upshock.