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Related Experiment Videos

A genome-wide screen for site-specific DNA-binding proteins.

Tony R Hazbun1, Stanley Fields

  • 1Department of Genome Sciences, Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98195, USA. thazbun@u.washington.edu

Molecular & Cellular Proteomics : MCP
|September 20, 2002
PubMed
Summary

This study developed a biochemical genomics method to identify proteins that bind DNA regulatory elements. The method identified Rgt1 as a novel transcription factor binding to the SUC2 gene promoter, impacting invertase activity.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Biochemistry

Background:

  • Identifying proteins that bind to specific DNA sequences is crucial for understanding gene regulation.
  • Existing methods can be time-consuming and may not cover a large portion of an organism's proteome.

Purpose of the Study:

  • To develop and validate a high-throughput biochemical genomics approach for identifying DNA-binding proteins.
  • To identify novel transcription factors that bind to the upstream activation sequence (UAS) of the Saccharomyces cerevisiae SUC2 gene.

Main Methods:

  • Utilized a nearly complete set of glutathione S-transferase (GST) fusion proteins from Saccharomyces cerevisiae.
  • Assayed protein binding to the SUC2 gene UAS using a biochemical genomics method.
  • Employed gel shift assays to characterize DNA-binding activities and identify specific binding sites.

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  • Assessed the in vivo role of identified transcription factors by analyzing gene expression in knockout strains.

    • Main Results:

    • The method successfully identified specific and nonspecific DNA-binding activities.
    • Three transcription factors, Mig1, Yer028c, and Rgt1, were identified as specific binders to the SUC2 UAS.
    • Mig1 and Yer028c binding validated the method's accuracy.
    • Rgt1 was identified as a novel SUC2-binding factor, with its binding site localized to the SUC2-B element.
    • Deletion of RGT1 in vivo led to reduced SUC2 gene expression under inducing conditions.

    Conclusions:

    • The developed biochemical genomics approach is effective for rapidly identifying DNA-binding proteins across an organism's proteome.
    • Rgt1 plays a role in the activated transcription of the SUC2 gene.
    • This method provides a powerful tool for discovering novel regulators of gene expression.