An intrinsically disordered transcription activation domain alters the DNA binding affinity and specificity of NFκB p50/RelA

Abstract

Many transcription factors contain intrinsically disordered transcription activation domains (TADs), which mediate interactions with co-activators 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. We biophysically characterized the NFκB p50/RelA heterodimer including the RelA TAD and investigated the influence of the TAD on NFκB-DNA interactions. In solution the RelA TAD is disordered but compact, with helical tendency in two regions that interact with co-activators. 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. 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 increased the affinity for non-specific DNA sequences by over 10-fold, leading to an overall decrease in specificity for κB DNA sequences. Our results reveal a novel function of the RelA TAD in promoting binding to non-consensus DNA previously observed by in vivo studies of NFκB-DNA binding in response to strong inflammatory signals.

Transcription factors perform a 2-step search of the nucleus

“…I use the term IDR to refer to regions described as the 'non-DBD' by Brodsky et al 2020. Classically, it was argued that DNA binding domains and activation domains were independent, modular components, but this idea is approaching the end of its usefulness. In the few cases that have been carefully examined, activation domains can modulate DNA affinity, increase specificity for cognate motifs, or increase affinity for random DNA (Liu et al 2008;Krois et al 2018;Baughman et al 2022). For the remainder of this piece, I assume that true modularity is rare.…”