Abstract
TATA-binding protein (TBP) is a required factor in eukaryotic transcription. It specifically binds to an A-T-rich sequence of the promoter known as the TATA-box, and further recruits other core transcription factors to form the pre-initiation complex. Therefore, understanding the mechanism of TBP regulation will help in the study of gene expression. It has been known that the Modifier of Transcription1 (Mot1), a member of the Swi2/Snf2 ATPase family, is capable of displacing TBP from DNA using ATP hydrolysis. To study the mechanism of this displacement using single-molecule techniques, I fluorescently labeled yeast TBP using a sortase-mediated reaction and observed the kinetics of the Mot1-mediated TBP-DNA interaction in the purified system. The single-molecule experiments were able to distinguish between TBP interactions with the TATA-box and with the non-specific DNA on the same DNA molecule. In the absence of ATP, Mot1 substantially increased the TBP dwell time on the TATA-box without affecting the stability of TBP-non-specific DNA complexes, while in the presence of ATP, Mot1 kinetically destabilized both TBP-TATA-box and TBP-non-specific DNA complexes. These results give insight into the mechanism of Mot1 function.