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首页> 外文期刊>Journal of Molecular Biology >Purification and in vitro activities of the Bacillus subtilis TnrA transcription factor.
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Purification and in vitro activities of the Bacillus subtilis TnrA transcription factor.

机译：枯草芽孢杆菌TnrA转录因子的纯化和体外活性。

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摘要

The Bacillus subtilis nitrogen regulatory protein TnrA was purified and its interaction with the nrgAB regulatory region examined. The TnrA protein activates transcription from the nrgAB promoter in vitro. DNase I footprinting and methylation protection experiments demonstrated that TnrA binds to an inverted repeat, upstream of the -35 region of the nrgAB promoter. Gel mobility retardation assays were used to determine the affinity of TnrA for its DNA-binding site. The equilibrium dissociation binding constant for the interaction of TnrA with the nrgAB promoter fragment was 7.7 nM under the conditions used here. Mutations in the TnrA consensus sequence that reduce nrgAB expression in vivo were found to reduce significantly the in vitro affinity for TnrA. An A+T rich region located upstream of the TnrA-binding site was found to be necessary for optimal transcriptional activation. A mutant protein, TnrA(HTH), was constructed in which the putative helix-turn-helix DNA-binding motif was altered by exchanging two arginine residues for alanine residues. The TnrA(HTH) protein was unable to activate the in vivo expression of nrgAB and had an in vitro affinity for the nrgAB promoter that was significantly lower than that of the wild-type protein. Copyright 2000 Academic Press.
机译：纯化枯草芽孢杆菌氮调节蛋白TnrA，并检查其与nrgAB调节区的相互作用。 TnrA蛋白在体外激活nrgAB启动子的转录。 DNase I足迹和甲基化保护实验表明，TnrA与nrgAB启动子-35区域上游的反向重复序列结合。凝胶迁移阻滞测定法用于确定TnrA对其DNA结合位点的亲和力。在此处使用的条件下，TnrA与nrgAB启动子片段相互作用的平衡解离结合常数为7.7 nM。发现在体内降低nrgAB表达的TnrA共有序列中的突变显着降低了体外对TnrA的亲和力。发现位于TnrA结合位点上游的富含A + T的区域对于最佳转录激活是必需的。构建了突变蛋白TnrA（HTH），其中通过将两个精氨酸残基交换为丙氨酸残基来改变推定的螺旋-转-螺旋DNA结合基序。 TnrA（HTH）蛋白无法激活nrgAB的体内表达，并且对nrgAB启动子的体外亲和力明显低于野生型蛋白。版权所有2000学术出版社。

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