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Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation
12:54

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Published on: March 7, 2018

Transient-state kinetic analysis of transcriptional activator·DNA complexes interacting with a key coactivator

Amberlyn M Wands1, Ningkun Wang, Jenifer K Lum

  • 1Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.

The Journal of Biological Chemistry
|February 15, 2011
PubMed

Insights

Transcriptional activators like Gal4, Gcn4, and VP16 recruit the coactivator Med15 through a shared binding mechanism. This process involves a conformational change in the Med15·activator·DNA complex, regulating transcriptional activation.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Prototypical amphipathic transcriptional activators (Gal4, Gcn4, VP16) interact with the coactivator Med15 (Gal11) during transcription initiation.
  • Evidence suggests these activators utilize a shared binding surface on Med15.
  • Little sequence homology exists among these activators, prompting investigation into their binding mechanism.

Purpose of the Study:

  • To determine if Gal4, Gcn4, and VP16 share a common binding mechanism for Med15 recruitment.
  • To characterize the thermodynamics and kinetics of Med15·activator·DNA complex formation.
  • To correlate binding kinetics with transcriptional activation potency.

Main Methods:

  • Fluorescence titration to study equilibrium binding.
  • Stopped-flow techniques to analyze reaction kinetics.
  • Characterization of Med15·activator·DNA complex formation.

Main Results:

  • Med15 recruitment by each activator followed a biphasic time course, indicating a two-step binding mechanism.
  • The mechanism involves a diffusion-limited bimolecular association followed by a conformational change.
  • The equilibrium constant for the conformational change (K(2)) directly correlates with activator potency (VP16 > Gal4 > Gcn4).

Conclusions:

  • Transcriptional activation is regulated, at least in part, by conformational rearrangements within the Med15·activator·DNA ternary complex.
  • The study provides the first detailed kinetic characterization of the transcriptional activation machinery.
  • Findings offer a framework for designing more potent transcriptional activators.

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