Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

Kinetic preference for oriented DNA binding by the yeast TATA-binding protein TBP.

Y Liu1, A Schepartz

  • 1Department of Chemistry, Yale University, New Haven, Connecticut 06520-8107, USA.

Biochemistry
|May 24, 2001
PubMed
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.

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Imaging organelle membranes in live cells at the nanoscale with lipid-based fluorescent probes.

Current opinion in chemical biology·2021
Same author

Effects of nucleic acids and polyanions on dimer formation and DNA binding by bZIP and bHLHZip transcription factors.

Bioorganic & medicinal chemistry·2001
Same author

Electrostatic control of half-site spacing preferences by the cyclic AMP response element-binding protein CREB.

Nucleic acids research·2001
Same author

Concerted evolution of structure and function in a miniature protein.

Journal of the American Chemical Society·2001
Same author

Methodology for optimizing functional miniature proteins based on avian pancreatic polypeptide using phage display.

Bioorganic & medicinal chemistry letters·2001
Same author

Hepatitis B virus protein pX enhances the monomer assembly pathway of bZIP.DNA complexes.

Biochemistry·2001

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.

Related Experiment Videos

  • 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.