Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California, USA.
Department of Chemistry and Center for Theoretical Biological Physics, Rice University, Houston, Texas, USA.
J Biol Chem. 2022 Sep;298(9):102349. doi: 10.1016/j.jbc.2022.102349. Epub 2022 Aug 5.
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.
许多转录因子含有固有无序的转录激活结构域（TAD），它们与共激活因子相互作用以激活转录。历史上，DNA 结合结构域和 TAD 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的 TAD 尚不清楚。在这里，我们从生物物理角度表征了包括 RelA TAD 的 NFκB p50/RelA 异源二聚体，并研究了 TAD 对 NFκB-DNA 相互作用的影响。在溶液中，我们表明 RelA TAD 是无序的，但紧凑的，在与共激活因子相互作用的两个区域具有螺旋倾向。我们确定 TAD 的存在通过促进 NFκB 二聚体结合超过κB 位点的数量，增加了含有串联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，我们测量了 p50/RelA 与含有串联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 序列的整体特异性降低。相比之下，先前的研究一般报告 TADs 降低 DNA 结合亲和力并提高序列特异性。我们的结果揭示了 RelA TAD 在促进与非共识 DNA 结合方面的新功能，这为 NFκB 在强炎症信号响应中体内广泛非共识 DNA 结合的先前观察结果提供了启示。
