The salt dependence of DNA recognition by NF-κB p50:: a detailed kinetic analysis of the effects on affinity and specificity
The salt dependence of DNA recognition by NF-κB p50:: a detailed kinetic analysis of the effects on affinity and specificity
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DOI：
10.1093/nar/27.4.1063
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发表时间：
1999-02-15
影响因子：
14.9
通讯作者：
Blackburn, JM
中科院分区：
文献类型：
--
作者：
Hart, DJ;Speight, RE;Blackburn, JM
使用表面等离子体共振生物传感器，在不同氯化钾浓度条件下测定NF-κ B p50与Ig-κ B位点和与无特异性结合位点的DNA双链体的结合动力学。测量结合和解离速率常数，从而能够计算解离常数。在先前建立的高亲和力缓冲液条件下，两个序列的k（a）为10（7）M（-1）s（-1）的数量级，而k（d）值在10（-1）和10（-4）s（-1）之间以序列依赖性方式变化600倍，表明p50对不同序列的选择性主要通过序列依赖性解离速率介导，计算的Ig-κ B序列的K-D值为16 pM，而非特异性序列的K-D为9.9 nM。随着离子强度增加到更接近细胞环境的水平，p50与非特异性序列的结合被消除，而特异性亲和力下降到纳摩尔水平。根据这些结果，提出了一种机制，其中p50以高亲和力结合特异性序列，同时结合非特异性序列足够弱以允许有效搜索DNA。
The binding kinetics of NF-kappa B p50 to the Ig-kappa B site and to a DNA duplex with no specific binding site were determined under varying conditions of potassium chloride concentration using a surface plasmon resonance biosensor. Association and dissociation rate constants were measured enabling calculation of the dissociation constants. Under previously established high affinity buffer conditions, the k(a) for both sequences was in the order of 10(7) M(-1)s(-1) whilst the k(d) values varied 600-fold in a sequence-dependent manner between 10(-1) and 10(-4) s(-1), suggesting that the selectivity of p50 for different sequences is mediated primarily through sequence-dependent dissociation rates, The calculated K-D value for the Ig-kappa B sequence was 16 pM, whilst the K-D for the non-specific sequence was 9.9 nM. As the ionic strength increased to levels which are closer to that of the cellular environment, the binding of p50 to the non-specific sequence was abolished whilst the specific affinity dropped to nanomolar levels. From these results, a mechanism is proposed in which p50 binds specific sequences with high affinity whilst binding non-specific sequences weakly enough to allow efficient searching of the DNA.