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Kinetic preference for oriented DNA binding by the yeast TATA-binding protein TBP
1Department of Chemistry, Yale University, New Haven, Connecticut 06520-8107, USA.
Biochemistry
|May 24, 2001
Summary
Yeast TATA box binding protein (yTBP) binds DNA in two orientations. A faster association rate for the crystal structure orientation explains the observed equilibrium preference, confirming free energy differences.
Area of Science:
- Molecular Biology
- Biophysics
- Structural Biology
Background:
- TATA box binding protein (TBP) is crucial for transcription initiation in eukaryotes.
- In solution, yeast TBP (yTBP) exhibits dual orientational binding to promoter DNA.
- Approximately 60% of yTBP-DNA complexes adopt a crystal-observed orientation at equilibrium.
Purpose of the Study:
- To investigate the association kinetics of the two yTBP-DNA orientational isomers.
- To determine the contribution of binding kinetics to the equilibrium distribution of yTBP orientations.
- To confirm the free energy difference between the two orientational binding states.
Main Methods:
- Stopped-flow fluorescence resonance energy transfer (FRET) was employed.
- FRET monitored binding between engineered tryptophan in yTBP and aminocoumarin on DNA.
- Simultaneous, independent kinetic measurements of both binding orientations were achieved.
Main Results:
- The study confirmed the free energy difference between the two yTBP-DNA orientational isomers.
- Faster association kinetics were observed for the crystal structure orientation of yTBP binding.
- Equilibrium binding preference is driven by the kinetic association rate, not solely thermodynamic stability.
Conclusions:
- The orientational preference of yTBP on promoter DNA is kinetically controlled.
- Faster association in the crystal structure orientation dictates the equilibrium distribution.
- Understanding TBP binding kinetics provides insights into transcription initiation regulation.
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