DNA binding of purified transcription factor NF-kappa B. Affinity, specificity, Zn2+ dependence, and differential half-site recognition.
DNA binding of purified transcription factor NF-kappa B. Affinity, specificity, Zn2+ dependence, and differential half-site recognition.
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纯化转录因子 NF-kappa B 的 DNA 结合。亲和力、特异性、Zn2 依赖性和差异半位点识别。
DOI：
10.1016/s0021-9258(18)52428-5
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发表时间：
1991
期刊：
影响因子：
--
通讯作者：
P. Baeuerle
中科院分区：
文献类型：
--
作者：
U. Zabel;R. Schreck;P. Baeuerle
利用NF-κ B.DNA复合物对嵌入剂氯喹的不敏感性，从人胎盘细胞质中快速纯化NF-κ B转录因子。纯化的NF-κ B需要100 mM KCl或NaCl和7.5的pH以最佳地结合DNA。在不存在竞争者DNA的情况下，在小于5分钟内达到结合平衡，并且在0.1 mg/ml poly（dI-dC）的存在下，在1小时后达到结合平衡。NF-κ B的DNA结合被螯合剂1，10-邻菲咯啉特异性阻断，并且只能通过添加Zn 2+来重建。在最佳结合条件下，纯化的NF-κ B与其最常见的同源DNA基序5 '-GGGACTTTCC-3'的复合物的解离常数在10（-12）至10（-13）M的范围内。NF-κ B以较低的亲和力识别各种其他顺式作用κ B基序。已知的NF-κ B结合位点的比较分析和与仅包含一个半位点或单链κ B基序的合成多核苷酸和寡核苷酸的竞争实验表明，NF-κ B蛋白复合物中的两个DNA结合单体可以与十聚体同源基序的半位点差异地相互作用。
A rapid purification procedure for the NF-kappa B transcription factor from the cytosol of human placenta is demonstrated which exploits the insensitivity of the NF-kappa B.DNA complex toward the intercalating agent chloroquine. Purified NF-kappa B required 100 mM KCl or NaCl and a pH of 7.5 to optimally bind to DNA. Equilibrium of binding was reached within less than 5 min in the absence of competitor DNA and after 1 h in the presence of 0.1 mg/ml poly(dI-dC). DNA binding of NF-kappa B was specifically blocked by the chelating agent 1,10-orthophenantroline and could only be reconstituted by addition of Zn2+. Under optimal binding conditions, the dissociation constant for the complex of the purified NF-kappa B with its most frequent cognate DNA motif 5‘-GGGACTTTCC-3‘ was in the range of 10(-12) to 10(-13) M. Various other cis-acting kappa B motifs were recognized by NF-kappa B with lower affinities. A comparative analysis of known NF-kappa B-binding sites and competition experiments with synthetic polynucleotides and oligonucleotides encompassing only one half-site or single-stranded kappa B motifs suggested that the two DNA-binding monomers in the NF-kappa B protein complex can interact differentially with the half-sites of the decameric cognate motif.