ESCHERICHIA-COLI FIS PROTEIN ACTIVATES RIBOSOMAL-RNA TRANSCRIPTION INVITRO AND INVIVO
ESCHERICHIA-COLI FIS PROTEIN ACTIVATES RIBOSOMAL-RNA TRANSCRIPTION INVITRO AND INVIVO
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DOI：
10.1002/j.1460-2075.1990.tb07586.x
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发表时间：
1990-11-01
期刊：
影响因子：
11.4
通讯作者：
GOURSE, RL
中科院分区：
文献类型：
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作者：
ROSS, W;THOMPSON, JF;GOURSE, RL
上游激活区（UAR）有助于大肠杆菌核糖体RNA启动子rrnB P1的极高活性，使其活性比缺乏UAR的相同启动子增加20- 30倍。我们已经使用DNA酶fooprinting定义三个特定的网站在rrnB P1 UAR结合Fis，一种蛋白质，以前确定其在重组增强子功能在其他系统中的作用。我们发现，纯化的Fis激活转录启动子含有这些网站在体外10- 20倍，在浓度与这些网站的填充。三种方法表明，Fis有助于体内UAR的功能。首先，随着Fis结合位点的缺失，rrnB P1-lacZ融合物的活性逐渐丧失。第二，在Fis结合位点具有突变的rrnB P1启动子在体内具有5倍降低的转录活性，在体外显著降低Fis结合，并且在体外不显示Fis依赖性转录激活。第三，上游激活在fis-菌株中减少了5倍.我们发现，rRNA启动子在体内对Fis缺失的反应与rRNA调控的负反馈模型的预测雅阁。我们发现，fis是不是必不可少的两个控制系统的功能，已知调节rRNA，生长速率依赖性控制和严格控制。在这些结果的基础上，我们提出了Fis和上游激活系统在rRNA合成中的作用。
An upstream activation region (UAR) contributes to the extremely high activity of the Escherichia coli ribosomal RNA promoter, rrnB P1, increasing its activity 20- to 30-fold over that of the same promoter lacking the UAR. We have used DNase fooprinting to define three specific sites in the rrnB P1 UAR that bind Fis, a protein identified previously by its role in recombinational enhancer function in other systems. We find that purified Fis activates transcription from promoters containing these sites 10- to 20-fold in vitro at concentrations correlating with the filling of these sites. Three approaches indicate that Fis contributes to the function of the UAR in vivo. First, there is a progressive loss in the activity of rrnB P1-lacZ fusions as Fis binding sites are deleted. Second, an rrnB P1 promoter with a mutation in a Fis binding site has 5-fold reduced transcription activity in vivo, dramatically reduced Fis binding in vitro, and shows no Fis dependent transcription activation in vitro. Third, upstream activation is reduced 5-fold in a fis- strain. We show that rRNA promoters derepress in response to the loss of Fis in vivo in accord with the predictions of the negative feedback model for rRNA regulation. We find that fis is not essential for the function of two control systems known to regulate rRNA, growth rate dependent control and stringent control. On the basis of these results, we propose roles for Fis and the upstream activation system in rRNA synthesis.