Su W, Porter S, Kustu S, Echols H
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Proc Natl Acad Sci U S A. 1990 Jul;87(14):5504-8. doi: 10.1073/pnas.87.14.5504.
The NtrC protein activates transcription of the glnA operon of enteric bacteria by stimulating the formation of stable "open" complexes by RNA polymerase (sigma 54-holoenzyme form). To regulate the glnA promoter, NtrC binds to sites that have the properties of transcriptional enhancers: the sites will function far from the promoter and in an orientation-independent fashion. To investigate the mechanism of enhancer function, we have used electron microscopy to visualize the interactions of purified NtrC and RNA polymerase with their DNA binding sites and with each other. Under conditions that allow the formation of open complexes, about 30% of DNA molecules carry both RNA polymerase and NtrC bound to their specific sites. Of these, about 15% form looped structures in which NtrC and the RNA polymerase-promoter complex are in contact. The length of the looped DNA is that predicted from the spacing that was engineered between the enhancer and the glnA promoter (390 base pairs). As expected for activation intermediates, the looped structures disappear when RNA polymerase is allowed to transcribe the DNA. We conclude that the NtrC enhancer functions by means of a direct association between DNA-bound NtrC and RNA polymerase (DNA-looping model). Association of DNA-bound proteins appears to be the major mechanism by which different types of site-specific DNA transactions are localized and controlled.
NtrC蛋白通过刺激RNA聚合酶（σ54-全酶形式）形成稳定的“开放”复合物，来激活肠道细菌谷氨酰胺合成酶操纵子（glnA operon）的转录。为了调控glnA启动子，NtrC会结合到具有转录增强子特性的位点上：这些位点在远离启动子的位置也能发挥作用，且与方向无关。为了研究增强子功能的机制，我们利用电子显微镜观察了纯化的NtrC和RNA聚合酶与其DNA结合位点以及它们彼此之间的相互作用。在允许形成开放复合物的条件下，约30%的DNA分子同时结合了RNA聚合酶和NtrC到它们各自的特定位点上。其中，约15%形成了环状结构，NtrC与RNA聚合酶-启动子复合物相互接触。环状DNA的长度与在增强子和glnA启动子之间设计的间隔（390个碱基对）所预测的长度一致。正如激活中间体所预期的那样，当RNA聚合酶转录DNA时，环状结构会消失。我们得出结论，NtrC增强子通过DNA结合的NtrC与RNA聚合酶之间的直接结合发挥作用（DNA环化模型）。DNA结合蛋白的结合似乎是不同类型位点特异性DNA作用得以定位和控制的主要机制。
