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High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy
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Simple biochemical features underlie transcriptional activation domain diversity and dynamic, fuzzy binding to

Adrian L Sanborn1,2, Benjamin T Yeh2, Jordan T Feigerle1

  • 1Department of Structural Biology, Stanford University School of Medicine, Stanford, United States.

Elife
|April 27, 2021
PubMed

Insights

Researchers identified 150 new transcriptional activation domains (ADs) in yeast. Most ADs bind the Med15 protein, explaining how gene activators function and offering a new tool for protein engineering.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Gene activator proteins possess DNA-binding and transcriptional activation domains (ADs).
  • The structure and function of most ADs remain poorly understood, hindering a comprehensive understanding of gene regulation.

Purpose of the Study:

  • To systematically identify and characterize novel transcriptional activation domains (ADs) in yeast.
  • To elucidate the molecular mechanisms underlying AD function and their interaction with the Mediator complex.

Main Methods:

  • Systematic screening of yeast transcription factor domains for in vivo activation.
  • mRNA display assays to quantify AD-Med15 subunit interactions.
  • Structural modeling to analyze AD-Med15 binding interfaces.
  • Machine learning (neural network) to predict ADs in human and yeast proteomes.

Main Results:

  • 150 novel ADs were identified in yeast.
  • 73% of identified ADs bound the Med15 subunit of the Mediator complex, with binding strength correlating to activation.
  • AD-Med15 interactions are dynamic and characterized by 'fuzzy' binding, lacking specific sequence motifs but exhibiting biochemical constraints.
  • A neural network successfully predicted ADs across species and protein types.

Conclusions:

  • This study resolves the enigma of AD structure and function, revealing a conserved interaction mechanism with Mediator.
  • The findings provide a framework for understanding gene activation and offer predictive power for identifying functional domains in diverse proteomes.
  • The identified ADs and their binding properties can inform future research in gene regulation and protein engineering.
Keywords:
S. cerevisiaeactivation domainchromosomescomputational biologygene expressionhigh-throughput screeninghumanmachine learningmediator complexmolecular simulationsystems biologytranscription factor

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