Transcription factors ERα and Sox2 have differing multiphasic DNA and RNA binding mechanisms

Abstract

Many transcription factors (TFs) have been shown to bind RNA, leading to open questions regarding the mechanism(s) of this RNA binding and its role in regulating TF activities. Here we use biophysical assays to interrogate the kon, koff, and Kd for DNA and RNA binding of two model human transcription factors, ER and Sox2. Unexpectedly, we found that both proteins exhibited multiphasic nucleic acid binding kinetics. We propose that Sox2 RNA and DNA multiphasic binding kinetics could be explained by a conventional model for sequential Sox2 monomer association and dissociation. In contrast, ER nucleic acid binding exhibited biphasic dissociation paired with novel triphasic association behavior, where two apparent binding transitions are separated by a 10-20 min "lag" phase depending on protein concentration. We considered several conventional models for the observed kinetic behavior, none of which adequately explained all the ER nucleic acid binding data. Instead, simulations with a model incorporating sequential ER monomer association, ER nucleic acid complex isomerization, and product "feedback" on isomerization rate recapitulated the general kinetic trends for both ER DNA and RNA binding. Collectively, our findings reveal that Sox2 and ER bind RNA and DNA with previously unappreciated multiphasic binding kinetics, and that their reaction mechanisms differ with ER binding nucleic acids via a novel reaction mechanism.