Contributions of promoter context and structure to regulated expression of the F plasmid traY promoter in Escherichia coli K-12.
Contributions of promoter context and structure to regulated expression of the F plasmid traY promoter in Escherichia coli K-12.
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启动子背景和结构对大肠杆菌 K-12 中 F 质粒 traY 启动子调节表达的贡献。
DOI：
10.1111/j.1365-2958.1993.tb01577.x
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发表时间：
1993
影响因子：
3.6
通讯作者：
Silverman,PM
中科院分区：
文献类型：
--
作者：
Gaudin,HM;Silverman,PM
F质粒traY启动子在体内的表达需要两种宿主（E. coli）和质粒编码的蛋白质。通过转录本大小和引物延伸分析判断，F质粒traY启动子在体外被纯化的E. coliσ; 70 RNA聚合酶。然而，在体外转录需要超螺旋模板。核酸内切酶保护实验表明，RNA聚合酶不能在线性或松弛的环状模板中在traY启动子处形成稳定的复合物，在体外，线性模板的转录可以通过改变traY-10和-35六聚体的共有序列来引发。在体外降低模板超螺旋对表观启动子转录的影响的改变也降低了F质粒TraJ蛋白对TraJ蛋白在体内表达的影响。在模板竞争实验中估计的表观TraJ启动子转录在体外不受启动子上游的traDNA缺失的影响，这是一种启动子环境的改变，在TraJ−细胞中引起高水平的启动子活性。这些数据提示了一种调控traY启动子活性的模型，其中涉及traDNA的核蛋白复合物直接位于traY启动子DNA的上游，使其局部松弛。TraJ和可能的其他激活剂可以破坏复合物，使启动子DNA在普遍的负超螺旋密度下平衡，从而引发转录起始。
Expression of the F plasmidtraYpromoterin vivorequires both host(E. coli)and plasmid encoded proteins. As judged by transcript size and primer extension analyses, the F plasmidtraYpromoter was utilizedin vitroby purifiedE. coliσ;70RNA polymerase in the absence of other proteins. However,in vitrotranscription required supercoiled templates. Endonuclease protection experiments showed that RNA polymerase is unable to form a stable complex at thetraYpromoter in linear or relaxed circular templates,in vitrotranscription with linear templates could be elicited by altering thetraY‐10 and −35 hexamers to the consensus sequences. Alterations that reduced the effect of template supercoiling on apparent promoter strengthin vitroalso reduced the effect of the F plasmid TraJ protein ontraYexpressionin vivo.ApparenttraYpromoter strengthin vitro, estimated in template competition experiments, was unaltered by deletion oftraDNA normally upstream of the promoter, a change in promoter context that elicited high levels of promoter activity in TraJ−cells. These data suggest a model for regulatedtraYpromoter activity in which a nucleoprotein complex involvingtraDNA immediately upstream locally relaxestraYpromoter DNA. TraJ and perhaps other activators could disrupt the complex, allowing promoter DNA to equilibrate at the prevailing negative superhelical density and thereby eliciting transcription initiation.