Contreras A, Drummond M
AFRC Institute of Plant Science Research, University of Sussex, Brighton, UK.
Nucleic Acids Res. 1988 May 11;16(9):4025-39. doi: 10.1093/nar/16.9.4025.
We have constructed mutations in what we predict to be the DNA-recognition helix of Klebsiella pneumoniae NtrC, which regulates transcription from promoters under global nitrogen control. Mutations which disrupt the helix lead to complete loss of function. All point mutants tested were able to activate transcription from the sigma 54-dependent glnA promoter, but only those retaining some ability to recognise NtrC binding sites, as evidenced by their ability to repress the ntrB promoter and the upstream glnA promoter, were able to activate the nifL promoter. One mutant, which contained an amino acid substitution in the turn of the DNA-binding motif as well as in the recognition helix, suppressed mutations in the NtrC binding sites upstream from the nifL promoter, but only if both sites bore equivalent transitions. This confirms that the DNA-binding motif for this class of transcriptional activator has been correctly identified and suggests that binding of NtrC can be cooperative.
我们已在肺炎克雷伯菌NtrC的DNA识别螺旋区域构建了突变体，该区域调控全局氮控制下启动子的转录。破坏该螺旋的突变会导致功能完全丧失。所有测试的点突变体都能够激活依赖于σ⁵⁴的谷氨酰胺合成酶基因（glnA）启动子的转录，但只有那些保留了一定识别NtrC结合位点能力的突变体（通过它们抑制ntrB启动子和上游glnA启动子的能力得以证明），才能激活固氮酶基因（nifL）启动子。一个突变体在DNA结合基序的转角以及识别螺旋区域都含有氨基酸替换，它能抑制nifL启动子上游NtrC结合位点的突变，但前提是两个位点都发生了等效的转变。这证实了这类转录激活因子的DNA结合基序已被正确识别，并表明NtrC的结合可能具有协同性。
