One-Hybrid Systems f hor Detecting Protein-DNA Interactions

  • Mary Kate Alexander
  • Brenda D. Bourns
  • Virginia A. Zakian
Part of the Methods in Molecular Biology book series (MIMB, volume 177)


The yeast two-hybrid assay has proven useful for detecting protein-protein interactions. A variation on the theme can be used for finding proteins that interact with a particular DNA sequence. The one-hybrid assay, so far carried out only in Saccharomyces cerevisiae, in its simplest form (Fig. 1) consists of a DNA sequence of interest placed upstream of a reporter gene. The reporter gene can be either on a plasmid (1) or integrated into the chromosome (2). The protein or library being tested is cloned into a vector that expresses that protein fused to a transcription activation domain (TAD), the equivalent of the prey protein in a two-hybrid assay. This hybrid protein is expressed in the strain carrying the reporter gene. If the protein is able to interact with the sequence of interest, by either binding directly to the DNA or indirectly via interaction with a DNA-binding protein, transcription of the reporter gene is activated.
Fig. 1.

A generic one-hybrid assay. The DNA sequence of interest is placed upstream of a reporter gene with a minimal promoter, either on a plasmid or integrated into a chromosome. A hybrid protein consisting of a TAD fused to a protein that interacts with the target site is able to activate transcription of the reporter gene.


Fusion Protein Reporter Gene Activation Domain Telomeric Sequence Hybrid Protein 
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Copyright information

© Humana Press Inc. 2001

Authors and Affiliations

  • Mary Kate Alexander
    • 1
  • Brenda D. Bourns
    • 1
  • Virginia A. Zakian
    • 1
  1. 1.Lewis Thomas LabPrinceton UniversityPrinceton

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