Tagami H, Aiba H
Department of Molecular Biology, School of Science, Nagoya University, Japan.
Nucleic Acids Res. 1995 Feb 25;23(4):599-605. doi: 10.1093/nar/23.4.599.
The role of cAMP receptor protein (CRP) in transcription activation at the Escherichia coli lac promoter was investigated focusing on the steps after the formation of open complex. Although CRP binding to the lac DNA is stabilized in the ternary open complex, a high concentration of heparin dissociates CRP from the open complex without affecting the interaction between RNA polymerase and promoter, resulting in a binary complex. The release of CRP is directly shown by Western blotting and DNase I footprinting. The binary complex exhibits a slightly increased gel mobility compared to the ternary complex. The binary complex retains the characteristics of the open complex in footprinting pattern which is essentially identical with that of the open complex of the lac UV5 promoter. The binary complex is competent for transcription. These results indicate that CRP is not necessary for the maintenance of active open complex. In addition, the removal of CRP does not increase the production of abortive RNAs. We conclude that the contact between CRP and RNA polymerase is not essential for transcription activation after the formation of the open complex at the lac promoter. In other words, the role of CRP in the lac promoter is restricted to the steps up to the formation of open complex.
研究了环磷酸腺苷受体蛋白（CRP）在大肠杆菌乳糖启动子转录激活中的作用，重点关注开放复合物形成后的步骤。尽管CRP与乳糖DNA的结合在三元开放复合物中得以稳定，但高浓度的肝素会使CRP从开放复合物中解离，而不影响RNA聚合酶与启动子之间的相互作用，从而形成二元复合物。通过蛋白质免疫印迹法和DNase I足迹法直接证实了CRP的释放。与三元复合物相比，二元复合物的凝胶迁移率略有增加。二元复合物在足迹模式上保留了开放复合物的特征，这与乳糖UV5启动子开放复合物的足迹模式基本相同。二元复合物具有转录活性。这些结果表明，CRP对于维持活性开放复合物并非必需。此外，去除CRP并不会增加流产RNA的产生。我们得出结论，在乳糖启动子开放复合物形成后，CRP与RNA聚合酶之间的接触对于转录激活并非必不可少。换句话说，CRP在乳糖启动子中的作用仅限于开放复合物形成之前的步骤。
