BPS2026 - Dissecting the TATA box sequence using smFRET

TATA binding protein (TBP) is a key component of the eukaryotic transcription initiation machinery because TBP interaction with a TATA box sequence triggers the assembly of the preinitiation complex, marking the first step in gene transcription. Structural and biochemical studies have shown that, upon binding of TBP to the TATA box, the dsDNA bends sharply. In this study, we used single-molecule fluorescence resonance energy transfer (smFRET) to study the TBP-induced DNA bending of the double-stranded DNA (dsDNA) of the human H2B promoter. We labeled the dsDNA with donor-acceptor dye pairs and probed the binding of yeast TBP to the TATA box sequence via the FRET efficiency changes. When the consensus H2B promoter sequence is investigated in the presence of TBP, we observed a low FRET peak corresponding to DNA alone and a high FRET peak indicating TBP binding and DNA bending. To test whether TBP’s high affinity for the TATA box arises from multiple potential binding positions, we introduced different point mutations to the consensus sequence, blocking potential binding sites. We detected a distribution of populations with high FRET efficiency due to TBP binding despite the presence of mutations, highlighting the stability of the TBP-DNA complex. Nevertheless, some differences exist in the position of the FRET efficiency peaks of the mutated DNAs, indicating the TBP binds at different positions along the TATA box sequence. Overall, our observations reveal new insights into the mechanism of the interaction between TBP and the TATA box sequence of the H2B promoter on a molecular level. These findings establish a framework for further investigations: we are currently extending our approach to investigate the bending of DNA in the presence of transcription cofactors, to study the details of DNA bending using graphene-energy-transfer and extend our approach to three-color FRET.