Protein Cofactors Are Essential for High-Affinity DNA Binding by the Nuclear Factor κB RelA Subunit.
Protein Cofactors Are Essential for High-Affinity DNA Binding by the Nuclear Factor κB RelA Subunit.
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DOI：
10.1021/acs.biochem.8b00158
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发表时间：
2018-05-22
期刊：
影响因子：
2.9
通讯作者：
Ghosh G
中科院分区：
文献类型：
--
作者：
Mulero MC;Shahabi S;Ko MS;Schiffer JM;Huang DB;Wang VY;Amaro RE;Huxford T;Ghosh G
转录激活蛋白通常包含两个功能结构域：DNA结合结构域(DBD)和激活结构域(AD)，前者以序列特异性与DNA结合，后者的既定功能是招募RNA聚合酶。在这份报告中，我们发现纯化的重组核因子κB(NF-κB)RelA二聚体与特定的κB DNA结合的亲和力显著低于从活化细胞核提取液中相同二聚体的亲和力，提示额外的核辅因子可能促进RELA二聚体与DNA的结合。此外，重组的relA在体外生理盐浓度下以相对较低的亲和力结合DNA。添加P53或RPS3(核糖体蛋白S3)可使RelA：DNA结合亲和力提高50倍，具体取决于蛋白质和离子条件。这些辅因子蛋白不形成稳定的三元复合体，表明它们通过动态相互作用稳定RelA：DNA复合体。令人惊讶的是，即使在辅因子存在的情况下，relA-DBD单独在相同的溶液条件下也不能与DNA结合，这表明relA-AD在DNA结合中发挥了重要作用。RelA降低：在生理离子强度下的DNA结合表明，体内多个辅助因子可能同时作用以减轻电解质效应并稳定RelA：DNA复合体。总体而言，我们的观察表明，relA-AD和多个辅因子蛋白协同作用，启动rela-DBD并稳定细胞中relA：DNA复合体。我们的研究为核辅因子蛋白在NF-κB依赖的基因调控中提供了一种机制。
Transcription activator proteins typically contain two functional domains: a DNA binding domain (DBD) that binds to DNA with sequence specificity and an activation domain (AD) whose established function is to recruit RNA polymerase. In this report, we show that purified recombinant nuclear factor κB (NF-κB) RelA dimers bind specific κB DNA sites with an affinity significantly lower than that of the same dimers from nuclear extracts of activated cells, suggesting that additional nuclear cofactors might facilitate DNA binding by the RelA dimers. Additionally, recombinant RelA binds DNA with relatively low affinity at a physiological salt concentration in vitro. The addition of p53 or RPS3 (ribosomal protein S3) increases RelA:DNA binding affinity 2- to >50-fold depending on the protein and ionic conditions. These cofactor proteins do not form stable ternary complexes, suggesting that they stabilize the RelA:DNA complex through dynamic interactions. Surprisingly, the RelA-DBD alone fails to bind DNA under the same solution conditions even in the presence of cofactors, suggesting an important role of the RelA-AD in DNA binding. Reduced RelA:DNA binding at a physiological ionic strength suggests that multiple cofactors might be acting simultaneously to mitigate the electrolyte effect and stabilize the RelA:DNA complex in vivo. Overall, our observations suggest that the RelA-AD and multiple cofactor proteins function cooperatively to prime the RelA-DBD and stabilize the RelA:DNA complex in cells. Our study provides a mechanism for nuclear cofactor proteins in NF-κB-dependent gene regulation.
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影响因子：
64.8
作者：
Jin, Fulai;Li, Yan;Dixon, Jesse R.;Selvaraj, Siddarth;Ye, Zhen;Lee, Ah Young;Yen, Chia-An;Schmitt, Anthony D.;Espinoza, Celso A.;Ren, Bing
通讯作者：
Ren, Bing
影响因子：
7.3
作者：
Cheng CS;Feldman KE;Lee J;Verma S;Huang DB;Huynh K;Chang M;Ponomarenko JV;Sun SC;Benedict CA;Ghosh G;Hoffmann A
通讯作者：
Hoffmann A
DOI：
10.1038/nsb0198-67
发表时间：
1998-01-01
期刊：
NATURE STRUCTURAL BIOLOGY
影响因子：
--
作者：
Chen, YQ;Ghosh, S;Ghosh, G
通讯作者：
Ghosh, G
影响因子：
4.8
作者：
Mulero, Maria Carmen;Huang, De-Bin;Ghosh, Gourisankar
通讯作者：
Ghosh, Gourisankar
影响因子：
14.9
作者：
Lecoq L;Raiola L;Chabot PR;Cyr N;Arseneault G;Legault P;Omichinski JG
通讯作者：
Omichinski JG