Freeing the Promoter Site: Mechanistic Insights into the Interaction of Mot1 with TBP using spFRET

Abstract

Gene transcription is a central process of life and is highly regulated with a multitude of transcription factors. Recently, we could show that dynamics of the Tata-box Binding Protein (TBP) on the promoter site also plays a role in gene regulation. Using single-pair Forster Resonance Energy Transfer (spFRET) experiments, we have monitored the conformation of TBP bound to the H2B promoter site in the presence and absence of Mot1. Upon the binding of Mot1 to the TBP-DNA complex, a high FRET state between TBP and labeled DNA is formed. Contrary to what was expected, Mot1 bound to TBP-DNA complexes was unable to dissociate TBP from the DNA upon addition of ATP. Instead, the TBP-DNA complex returned to its original conformation even though Mot1 remain bound to the complex. Only when Mot1 was also present at nM concentrations in solution was dissociation of TBP from the promoter site observed upon addition of ATP. This suggests that a single Mot1 is either insufficient or inefficient at dissociating TBP alone.We also used spFRET to monitor the DNA conformation during the interaction of Mot1 with TBP-DNA. TBP-bound DNA was observed to fluctuate between three conformations. The FRET values of the conformation varied depending on the orientation of TBP on the promoter. Fluctuations between the same conformations were observed when Mot1 was bound to the TBP-DNA complex but with an altered kinetics. The results suggest that Mot1 primes incorrectly bound TBP for dissociation, preferably freeing promoter sites with an inappropriately bound TBP molecule.