Cannon W, Buck M
AFRC Institute of Plant Science Research, Nitrogen Fixation Laboratory, University of Sussex, Brighton, U.K.
J Mol Biol. 1992 May 20;225(2):271-86. doi: 10.1016/0022-2836(92)90921-6.
The positive control protein NifA of Klebsiella pneumoniae activates transcription by RNA polymerase containing sigma 54 by catalysing open promoter complex formation. We show that the integrity of the putative ATP-binding pocket in the central domain of NifA is necessary for the positive control function of NifA, but is not required for DNA-binding or recognition of NifA by NifL. The inactive mutant NifA proteins are trans dominant to wild-type NifA and are unable to catalyse formation of open promoter complexes irrespective of whether a closed promoter complex at the nifH promoter has preformed. Formation of the closed complex results in a DNA structural distortion adjacent to the DNA region melted in the open promoter complex. This distortion lies at the leading edge of the E sigma 54 footprint. Although unable to catalyse open complex formation, some mutant NifAs altered the chemical reactivity of the distorted base-pair indicating that they retain the ability to recognize the closed promoter complex. The activation phenotype of partially active NifA molecules was sensitive to promoter sequences known to influence closed complex formation, indicating differences in (1) the susceptibility of the closed complexes towards activation and (2) their requirements for NifA during activation.
肺炎克雷伯菌的阳性对照蛋白NifA通过催化开放启动子复合物的形成，激活含σ54的RNA聚合酶的转录。我们发现，NifA中央结构域中假定的ATP结合口袋的完整性对于NifA的阳性对照功能是必需的，但对于DNA结合或NifL对NifA的识别并非必需。无活性的突变型NifA蛋白对野生型NifA具有反式显性，并且无论nifH启动子处是否已形成封闭启动子复合物，都无法催化开放启动子复合物的形成。封闭复合物的形成导致与开放启动子复合物中解链的DNA区域相邻的DNA结构扭曲。这种扭曲位于Eσ54足迹的前沿。尽管一些突变型NifA无法催化开放复合物的形成，但它们改变了扭曲碱基对的化学反应性，表明它们保留了识别封闭启动子复合物的能力。部分活性NifA分子的激活表型对已知影响封闭复合物形成的启动子序列敏感，这表明（1）封闭复合物对激活的敏感性以及（2）它们在激活过程中对NifA的需求存在差异。
