Transcription factors of the nuclear factor (NF)-κB/Rel family translocate into the nucleus upon degradation of the IκBs. Postinduction repression of NF-κB activity depends on NF-κB–regulated resynthesis of IκBα, which dissociates NF-κB from DNA and exports it to the cytosol. We found that after activation, p65/RelA is degraded by the proteasome in the nucleus and in a DNA binding–dependent manner. If proteasome activity is blocked, NF-κB is not promptly removed from some target genes in spite of IκBα resynthesis and sustained transcription occurs. These results indicate that proteasomal degradation of p65/RelA does not merely regulate its stability and abundance, but also actively promotes transcriptional termination.

Because of its very high affinity for DNA, NF‐κB is believed to make long‐lasting contacts with cognate sites and to be essential for the nucleation of very stable enhanceosomes. However, the kinetic properties of NF‐κB interaction with cognate sites in vivo are unknown. Here, we show that in living cells NF‐κB is immobilized onto high‐affinity binding sites only transiently, and that complete NF‐κB turnover on active chromatin occurs in less than 30 s. Therefore, promoter‐bound NF‐κB is in dynamic equilibrium with nucleoplasmic dimers; promoter occupancy and transcriptional activity oscillate synchronously with nucleoplasmic NF‐κB and independently of promoter occupancy by other sequence‐specific transcription factors. These data indicate that changes in the nuclear concentration of NF‐κB directly impact on promoter function and that promoters sample nucleoplasmic levels of NF‐κB over a timescale of seconds, thus rapidly re‐tuning their activity. We propose a revision of the enhanceosome concept in this dynamic framework.