Cooperative action of separate interaction domains promotes high-affinity DNA binding of Arabidopsis thaliana ARF transcription factors

Mattia Fontana, Mark Roosjen, Isidro Crespo García, Willy van den Berg, Marc Malfois, Roeland Boer, Dolf Weijers*, Johannes Hohlbein*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

1 Citation (Scopus)


The signaling molecule auxin coordinates many growth and development processes in plants, mainly through modulating gene expression. Transcriptional response is mediated by the family of auxin response factors (ARF). Monomers of this family recognize a DNA motif and can homodimerize through their DNA-binding domain (DBD), enabling cooperative binding to an inverted binding site. Most ARFs further contain a C-terminal PB1 domain that is capable of homotypic interactions and mediating interactions with Aux/IAA repressors. Given the dual role of the PB1 domain, and the ability of both DBD and PB1 domain to mediate dimerization, a key question is how these domains contribute to DNA-binding specificity and affinity. So far, ARF–ARF and ARF–DNA interactions have mostly been approached using qualitative methods that do not provide a quantitative and dynamic view on the binding equilibria. Here, we utilize a DNA binding assay based on single-molecule Förster resonance energy transfer (smFRET) to study the affinity and kinetics of the interaction of several Arabidopsis thaliana ARFs with an IR7 auxin-responsive element (AuxRE). We show that both DBD and PB1 domains of AtARF2 contribute toward DNA binding, and we identify ARF dimer stability as a key parameter in defining binding affinity and kinetics across AtARFs. Lastly, we derived an analytical solution for a four-state cyclic model that explains both the kinetics and the affinity of the interaction between AtARF2 and IR7. Our work demonstrates that the affinity of ARFs toward composite DNA response elements is defined by dimerization equilibrium, identifying this as a key element in ARF-mediated transcriptional activity.

Original languageEnglish
Article numbere2219916120
Number of pages10
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number11
Publication statusPublished - 7 Mar 2023


  • auxin response factor
  • fourstate cyclic model
  • protein–DNA interaction
  • smFRET
  • transcription factor


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