Yeast One-Hybrid (Y1H) Assay: Principles, Systems, and Applications

Introduction to Yeast One-Hybrid (Y1H) Technology

The Yeast One-Hybrid (Y1H) assay was first developed by Li and colleagues in 1993, based on the principle of the Yeast Two-Hybrid (Y2H) system. Unlike Y2H, which detects protein–protein interactions, Y1H focuses on DNA–protein interactions (PDIs).

By leveraging the yeast genetic expression system, Y1H links the binding of transcription factors (TFs) or other DNA-binding proteins to the activation of a reporter gene, enabling systematic study of gene regulation in eukaryotic cells.

Core mechanism: A promoter or DNA motif of interest replaces the Gal4p-binding site in yeast. If the prey protein (fused with GAL4 activation domain) binds to this DNA sequence, the downstream reporter gene is activated. Theoretically, any regulatory DNA element can be used to capture interacting proteins.This makes Y1H not only useful for discovering transcriptional activators, but also for identifying repressors and proteins involved in DNA replication or transcriptional regulation.

Evolution of Y1H Systems

Early Y1H assays (e.g., Y187-pHis2) used episomal bait vectors. However, since promoters are naturally located in the genome, integration-based systems were later developed for greater physiological relevance.

• YM4271-pHisi-1 / pLacZi system: Integrated but prone to high false positives when using pLacZi alone.

• Y1HGold-pAbAi system: Currently the most widely used platform, balancing integration with reduced background activation.

Screening Strategies in Y1H

Two main approaches are widely adopted:

1. cDNA Library Screening

    a. Commonly used when TF libraries are unavailable.

    b. Captures DNA-binding proteins from a given species, including transcription regulators, histones, and nucleases.

2. Transcription Factor (TF) Library Screening

    a. Higher efficiency in identifying TFs.

    b. Published TF libraries include human, rice, and Arabidopsis.

    c. While TF libraries are powerful, they are costly to build.

In practice, many researchers still use promoter-centered Y1H with cDNA libraries as a cost-effective strategy.

Comparison of Screening Approaches

Frequently Asked Questions (FAQs)

1. What length of promoter fragment should be used for bait construction?

 There is no strict rule. Fragments can range from short motifs to full-length promoters.

2. How should bait sequences be selected?

• Promoter fragment approach: Focus on ~200–300 bp regions enriched in cis-elements.

• Motif approach: Use tandem repeats (×3) of a motif of interest.

 Useful promoter databases: PlantCARE, PlantPAN 3.0, PlantTFDB.

3. How to set negative and positive controls?

• Negative controls: Mutated motif (pAbAi-mutant) or Y1HGold(pAbAi-promoter) + pGADT7.

• Positive controls: Known TF–promoter pairs.

4. Is cell density adjustment necessary during autoactivation tests?

 Yes. For accurate results, dilute to OD ≈ 0.002 (~100–2000 colonies per 90 mm plate).

5. What if autoactivation cannot be suppressed?

 Test multiple promoter fragments (2–3 designs). Switching to another system rarely solves the issue.

6. Does Y1H guarantee identification of transcription factors?

 Not always. While TFs can be detected, their representation in cDNA libraries is limited.

• For higher success:Use rice TF libraries for monocots.

• Use Arabidopsis TF libraries for dicots.

 Validate candidate TFs with one-to-one interaction assays in the target species.

Why Choose Omics Empower Yeast One-Hybrid (Y1H) Service?

At Omics Empower, we provide end-to-end yeast hybrid solutions, including  Yeast One-Hybrid (Y1H), Yeast Two-Hybrid (Y2H), and custom library construction and screening. Our platform enables researchers to systematically study DNA–protein interactions and protein–protein interactions, supporting discoveries in gene regulation and molecular biology.

As one of the earliest companies to commercialize yeast hybrid technologies in Asia, Omics Empower has been a pioneer in Y1H and Y2H services. We were among the first to master both Gateway and SMART library construction methods, and we apply multiple yeast hybrid systems, including GAL4 and ubiquitin-based platforms, to meet the diverse needs of global research partners.

Backed by more than a decade of expertise, our yeast library platform has already supported 200+ customer publications, including high-impact studies featured on the covers of Science and Cell. This track record demonstrates both the reliability of our data and the trust placed in our services by the scientific community.

Proven Quality

• 10+ years of yeast library expertise with 200+ customer publications, including Science and Cell cover papers.

• Gateway and SMART construction methods to meet different project requirements.

• Rigorous QC: 12 checkpoints and 7 sequencing rounds guarantee reliability.

• High transformation efficiency (>10⁶ CFU), ensuring full representation.

Dedicated Support

• Expert technical team: molecular biologists, bioinformatics specialists, and local account managers.

• One year of free after-sales support, covering troubleshooting and data analysis.

Contact our team today to discuss your research needs and explore how our yeast library and screening services can help drive your next research.