Conformational changes and catalytic inefficiency associated with Mot1-mediated TBP-DNA dissociation

Area of Science:

• Molecular Biology
• Biochemistry
• Gene Regulation

Background:

• TATA-box Binding Protein (TBP) is crucial for gene expression and pre-initiation complex formation.
• Modifier of transcription 1 (Mot1) is a TBP-associated ATPase that regulates TBP's promoter occupancy.
• The precise molecular mechanism of Mot1's action on TBP-DNA complexes remains unclear.

Purpose of the Study:

• To elucidate the molecular mechanism by which Mot1 interacts with and influences the TBP-DNA complex.
• To investigate the role of ATP hydrolysis by Mot1 in TBP displacement.
• To understand how Mot1 activity is regulated for dynamic control of TBP levels at promoters.

Main Methods:

• Single-pair Förster resonance energy transfer (spFRET) was employed to study the dynamics of the DNA/TBP complex.
• spFRET measurements were performed on dual-labeled DNA to track TBP interactions.
• Adenosine triphosphate (ATP) hydrolysis was monitored in the presence of Mot1 and the DNA/TBP complex.

Main Results:

• Mot1 binding induced a conformational change in the DNA/TBP complex.
• ATP hydrolysis by Mot1 caused a conformational shift but did not lead to efficient TBP dissociation.
• spFRET data indicated that Mot1's ATPase activity primes TBP for dissociation, requiring additional Mot1 for complete unbinding.

Conclusions:

• Mot1's ATPase activity prepares TBP for removal, but does not solely cause dissociation.
• A two-step mechanism involving Mot1-mediated priming and subsequent Mot1 action is proposed for TBP unbinding.
• This mechanism allows for dynamic regulation of TBP promoter occupancy without disrupting essential TBP-DNA interactions.