Many transcription factors contain intrinsically disordered transcription activation domains (TADs), which mediate interactions with coactivators to activate transcription. Historically, DNA-binding domains and TADs have been considered as modular units, but recent studies have shown that TADs can influence DNA binding. Whether these results can be generalized to more TADs is not clear. Here, we biophysically characterized the NFκB p50/RelA heterodimer including the RelA TAD and investigated the TAD’s influence on NFκB–DNA interactions. In solution, we show the RelA TAD is disordered but compact, with helical tendency in two regions that interact with coactivators. We determined that the presence of the TAD increased the stoichiometry of NFκB–DNA complexes containing promoter DNA sequences with tandem κB recognition motifs by promoting the binding of NFκB dimers in excess of the number of κB sites. In addition, we measured the binding affinity of p50/RelA for DNA containing tandem κB sites and single κB sites. While the presence of the TAD enhanced the binding affinity of p50/RelA for all κB sequences tested, it also increased the affinity for nonspecific DNA sequences by over 10-fold, leading to an overall decrease in specificity for κB DNA sequences. In contrast, previous studies have generally reported that TADs decrease DNA-binding affinity and increase sequence specificity. Our results reveal a novel function of the RelA TAD in promoting binding to nonconsensus DNA, which sheds light on previous observations of extensive nonconsensus DNA binding by NFκB in vivo in response to strong inflammatory signals.

许多转录因子含有本质上无序的转录激活域（TAD），它介导与共激活因子的相互作用以激活转录。历史上，DNA 结合域和 TAD 一直被认为是模块化单元，但最近的研究表明 TAD 可以影响 DNA 结合。这些结果是否可以推广到更多 TAD 尚不清楚。在这里，我们对包括 RelA TAD 在内的 NFκB p50/RelA 异二聚体进行了生物物理表征，并研究了 TAD 对 NFκB-DNA 相互作用的影响。在解决方案中，我们表明 RelA TAD 是无序但紧凑的，在与共激活子相互作用的两个区域中具有螺旋趋势。我们确定，TAD 的存在通过促进超过 κB 位点数量的 NFκB 二聚体结合，增加了包含具有串联 κB 识别基序的启动子 DNA 序列的 NFκB-DNA 复合物的化学计量。此外，我们还测量了 p50/RelA 对含有串联 κB 位点和单个 κB 位点的 DNA 的结合亲和力。虽然 TAD 的存在增强了 p50/RelA 对所有测试的 κB 序列的结合亲和力，但它也使对非特异性 DNA 序列的亲和力增加了 10 倍以上，导致对 κB DNA 序列的特异性总体下降。相比之下，之前的研究普遍报道 TAD 会降低 DNA 结合亲和力并增加序列特异性。我们的结果揭示了 RelA TAD 在促进与非共有 DNA 结合方面的新功能，这为之前观察到的体内NFκB响应强炎症信号而广泛结合非共有 DNA 的观察结果提供了线索。