Institute of Physics, School of Basic Science, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.
Current address: EMBL Heidelberg, Germany.
PLoS Genet. 2019 Jan 17;15(1):e1007891. doi: 10.1371/journal.pgen.1007891. eCollection 2019 Jan.
Transcription factors (TFs) regulate gene expression in both prokaryotes and eukaryotes by recognizing and binding to specific DNA promoter sequences. In higher eukaryotes, it remains unclear how the duration of TF binding to DNA relates to downstream transcriptional output. Here, we address this question for the transcriptional activator NF-κB (p65), by live-cell single molecule imaging of TF-DNA binding kinetics and genome-wide quantification of p65-mediated transcription. We used mutants of p65, perturbing either the DNA binding domain (DBD) or the protein-protein transactivation domain (TAD). We found that p65-DNA binding time was predominantly determined by its DBD and directly correlated with its transcriptional output as long as the TAD is intact. Surprisingly, mutation or deletion of the TAD did not modify p65-DNA binding stability, suggesting that the p65 TAD generally contributes neither to the assembly of an "enhanceosome," nor to the active removal of p65 from putative specific binding sites. However, TAD removal did reduce p65-mediated transcriptional activation, indicating that protein-protein interactions act to translate the long-lived p65-DNA binding into productive transcription.
转录因子（TFs）通过识别和结合特定的 DNA 启动子序列来调节原核生物和真核生物的基因表达。在高等真核生物中，TF 与 DNA 的结合持续时间与下游转录输出之间的关系尚不清楚。在这里，我们通过活细胞单分子成像技术研究了转录激活因子 NF-κB（p65）的 TF-DNA 结合动力学和全基因组定量 p65 介导的转录，从而解决了这个问题。我们使用了突变体 p65，分别干扰其 DNA 结合结构域（DBD）或蛋白-蛋白转录激活结构域（TAD）。我们发现，p65-DNA 结合时间主要由其 DBD 决定，并且只要 TAD 完整，它就与转录输出直接相关。令人惊讶的是，TAD 的突变或缺失并没有改变 p65-DNA 结合的稳定性，这表明 p65 的 TAD 通常既不参与“增强子复合物”的组装，也不参与 p65 从假定的特定结合位点的主动去除。然而，TAD 的去除确实降低了 p65 介导的转录激活，这表明蛋白质-蛋白质相互作用将长寿的 p65-DNA 结合转化为有效的转录。
