Probing the Escherichia coli glnALG upstream activation mechanism in vivo.
Probing the Escherichia coli glnALG upstream activation mechanism in vivo.
复制标题
探究大肠杆菌glnALG上游体内激活机制。
DOI：
10.1073/pnas.85.23.8934
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发表时间：
1988
影响因子：
11.1
通讯作者：
Gralla,JD
中科院分区：
文献类型：
--
作者：
Sasse-Dwight,S;Gralla,JD
在激活条件下glnA调节区的体内“足迹”表明，三个最上游的激活序列结合细胞中的蛋白质NRI。这些部位的保护层一起跨越了七个连续的主凹槽中的六个，并位于同一个螺旋面上。E sigma 54在glnAp 2启动子下游大约60个碱基对处保护DNA的两个主要沟，并且主要在相对的螺旋面上。使用高锰酸钾探测体内开放复合物形成的实验表明，NRI是E sigma 54打开启动子DNA所绝对需要的。总之，硫酸二甲酯和高锰酸盐研究证实了[Reitzer，L. J.，埃罗河，郑，W. D、Abrams，S.一、罗斯坦，D. M.，亨特，T。P.，泰勒，B。& Magasanik，B.（1987）J. Bacteriol. 169，4279-4284]，E σ 54在体内以封闭复合物的形式占据glnAp 2启动子，即使在过量氮存在和NRI不存在的情况下。此外，转录激活的缓慢步骤被证明是下游启动子DNA中的NRI依赖性构象变化，这导致DNA解链。这些观察放置有趣的限制模型描述的机制，NRI激活转录glnAp 2在一定距离。
In vivo "footprints" of the glnA regulatory region under activating conditions demonstrate that the three most upstream activator sequences bind the protein NRI in the cell. Together, protections at these sites span six of seven consecutive major grooves and lie on the same helix face. E sigma 54 protects two major grooves of DNA approximately 60 base pairs downstream at the glnAp2 promoter and primarily on the opposite helix face. Experiments using potassium permanganate to probe open complex formation in vivo demonstrate that NRI is absolutely required for E sigma 54 to open the promoter DNA. Together, the dimethyl sulfate and permanganate studies verify [Reitzer, L. J., Bueno, R., Cheng, W. D., Abrams, S. A., Rothstein, D. M., Hunt, T. P., Tyler, B. & Magasanik, B. (1987) J. Bacteriol. 169, 4279-4284] that E sigma 54 occupies the glnAp2 promoter in a closed complex in vivo even in the presence of excess nitrogen and the absence of NRI. Furthermore, the slow step in transcriptional activation is shown to be an NRI-dependent conformational change in the downstream promoter DNA, which results in DNA melting. These observations place interesting restrictions on models describing the mechanism by which NRI activates transcription from glnAp2 at a distance.