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A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies
Published on: July 24, 2017
Gene-centered yeast one-hybrid assays
John S Reece-Hoyes1, Albertha J M Walhout
1University of Massachusetts Medical School, Worcester, MA, USA.
Methods in Molecular Biology (Clifton, N.J.)
|January 6, 2012
Summary
Gene-centered yeast one-hybrid (Y1H) assays identify transcription factor (TF)-DNA interactions. This robust method reveals which TFs bind specific DNA elements, aiding in understanding gene regulation.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Transcription is controlled by sequence-specific transcription factors (TFs) binding to DNA elements in regulatory regions.
- Identifying TF-DNA interactions is crucial for understanding gene regulation.
- Existing methods are either TF-centered or gene-centered.
Purpose of the Study:
- To describe and validate gene-centered yeast one-hybrid (Y1H) assays for identifying TF-DNA interactions.
- To demonstrate the robustness of Y1H for diverse DNA elements and TF families.
Main Methods:
- Gene-centered yeast one-hybrid (Y1H) assay.
- Cloning DNA fragments upstream of reporter genes in yeast.
- Introducing plasmids expressing TF-Gal4 activation domain (AD) fusion proteins.
- Detecting reporter gene activation upon TF-DNA interaction.
- Sequencing plasmids from activated colonies to identify bound TFs.
Main Results:
- Y1H assays successfully detect TF-DNA interactions.
- The method is robust across various DNA elements and TF families.
- Reporter activation indicates TF binding, irrespective of the TF's in vivo function (activator or repressor).
Conclusions:
- Gene-centered Y1H is a powerful tool for *ab initio* identification of TF-DNA binding.
- This method facilitates the discovery of novel TF-DNA interactions.
- Y1H assays provide a reliable approach to map TF binding sites.
