RelB and RelE of Escherichia coli form a tight complex that represses transcription via the ribbon-helix-helix motif in RelB.
RelB and RelE of Escherichia coli form a tight complex that represses transcription via the ribbon-helix-helix motif in RelB.
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DOI：
10.1016/j.jmb.2009.09.006
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发表时间：
2009-11-27
影响因子：
5.6
通讯作者：
Gerdes K
中科院分区：
文献类型：
--
作者：
Overgaard M;Borch J;Gerdes K
RelB是大肠杆菌relBE毒素-抗毒素位点编码的条带-螺旋-螺旋（RHH）抑制因子，与relle相互作用，从而抵消relle的mRNA裂解活性。此外，RelB二聚体抑制强relBE启动子，RelB的这种抑制作用被RelE增强；也就是说，RelE作为转录协同抑制因子发挥作用。RelB是一种Lon蛋白酶底物，激活relBE转录和激活RelE的mRNA裂解活性都需要Lon。在这里，我们描述了对relBE模型操纵子的转录控制重要的分子相互作用。通过对relB突变体的体内筛选，我们发现了relB n端RHH基序形成的dna结合域内重要氨基酸位置的多个核苷酸变化。通过凝胶转移试验、转录融合试验和RelB-DNA结构模型分析这些突变RHH蛋白子集的DNA结合，揭示了在操作员（relO） DNA中形成关键DNA -主干接触的氨基酸残基。relO的突变和足迹分析表明，RelB二聚体结合在DNA螺旋的同一面上，RHH基序识别在二部分结合位点内的4个6-bp重复序列。每个半位点之间的间距对于邻接结合的RelB二聚体之间的合作相互作用至关重要，这些二聚体由共抑制因子RelE稳定。RelB和RelE相互作用的动力学和化学计量学测量证实，这两种蛋白质形成了一个高亲和力的复合物，化学计量比为2:1。Lon在体外降解RelB，而RelE抑制了RelB的降解，这与RelE在体内保护RelB免受Lon蛋白水解的说法一致。
RelB, the ribbon–helix–helix (RHH) repressor encoded by the relBE toxin–antitoxin locus of Escherichia coli, interacts with RelE and thereby counteracts the mRNA cleavage activity of RelE. In addition, RelB dimers repress the strong relBE promoter and this repression by RelB is enhanced by RelE; that is, RelE functions as a transcriptional co-repressor. RelB is a Lon protease substrate, and Lon is required both for activation of relBE transcription and for activation of the mRNA cleavage activity of RelE. Here we characterize the molecular interactions important for transcriptional control of the relBE model operon. Using an in vivo screen for relB mutants, we identified multiple nucleotide changes that map to important amino acid positions within the DNA-binding domain formed by the N-terminal RHH motif of RelB. Analysis of DNA binding of a subset of these mutant RHH proteins by gel-shift assays, transcriptional fusion assays and a structure model of RelB–DNA revealed amino acid residues making crucial DNA–backbone contacts within the operator (relO) DNA. Mutational and footprinting analyses of relO showed that RelB dimers bind on the same face of the DNA helix and that the RHH motif recognizes four 6-bp repeats within the bipartite binding site. The spacing between each half-site was found to be essential for cooperative interactions between adjacently bound RelB dimers stabilized by the co-repressor RelE. Kinetic and stoichiometric measurements of the interaction between RelB and RelE confirmed that the proteins form a high-affinity complex with a 2:1 stoichiometry. Lon degraded RelB in vitro and degradation was inhibited by RelE, consistent with the proposal that RelE protects RelB from proteolysis by Lon in vivo.
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影响因子：
16
作者：
Kamada, K;Hanaoka, F
通讯作者：
Hanaoka, F
DOI：
10.1073/pnas.251327898
发表时间：
2001-12-04
影响因子：
11.1
作者：
Christensen, SK;Mikkelsen, M;Gerdes, K
通讯作者：
Gerdes, K
影响因子：
3.6
作者：
Christensen, SK;Gerdes, K
通讯作者：
Gerdes, K
影响因子：
16
作者：
Kamada, K;Hanaoka, F;Burley, SK
通讯作者：
Burley, SK
影响因子：
2.9
作者：
Cherny, Izhack;Overgaard, Martin;Gazit, Ehud
通讯作者：
Gazit, Ehud