Yeast Three-Hybrid (Y3H) Service

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Yeast Three-Hybrid (Y3H) Service

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CD BioSciences provides clients with yeast three-hybrid (Y3H) services that can analyze plant protein-RNA interactions. In the Y3H system, RNA-protein interactions lead to the transcription of reporter genes and can be detected by cell growth, clone color, or specific enzyme activity. Our service enables the resolution of advanced RNA-protein complexes and reveals the molecular mechanisms in plant life processes.

What is Y3H?

The Y3H system is a powerful tool for demonstrating RNA-protein interactions. The Y3H consists of three chimeric assemblies, chimeric proteins containing DNA and RNA-binding structural domains that link the RNA to the promoter of a reporter gene. This chimeric protein usually consists of a LexA/MS2 shell protein fusion. The transcription activation structural domain (AD) binds to the test protein. The reporter gene is activated when the RNA interacts with the test protein.

Schematic illustration of Y3H.   Figure 1. Schematic illustration of Y3H. (Wurster, S. E., & Maher, L. J., 3rd., 2010)

Service Content

Workflow

We are committed to providing one-stop Y3H services, the specific experiments steps include

  • Introduction of RNA plasmids and cDNA libraries.
  • Preparation of yeast sensory cells (LiAc method)
  • Transformation of plasmids/libraries
  • Removal of RNA-independent false positives by clone color.
  • β-Gal activity analysis.

To ensure that white clones containing cDNAs are activated by Y3H, we assay the expression level of the lacZ gene with the following techniques.

  • Qualitative (filter paper) assay
  • Quantitative (liquid) assay (ONPG method)
  • Purified RNA Plasmid re-assay.
  • Determination of binding specificity using mutant and control RNAs.
  • Identification of positive cDNAs.

Y3H service expansion

  • Tertiary structure and accessibility analysis of RNA insertions.
  • Determining the association between reporter gene activation and the strength of RNA-protein interactions.
  • Association between protein concentration and reporter gene expression.
  • Yeast strain improvement for Y3H experiments.

Sample Requirements

Sample types Details
Plant cell > 1x107
Plant tissue > 2g
Total RNA > 200µg

Technology Advantages

  • Y3H is performedin vivo and proteins are likely to retain the natural conformation.
  • The Y3H system does not require purification of target proteins and antibodies.
  • It is a sensitive method for detecting weak and transient interactions.
  • Wide range of applications, the third component can be a protein, RNA, or small molecule.
  • The action mode of the third component on the other two proteins (promotion or inhibition) can be characterized by the colony growth state.

CD BioSciences is committed to providing cutting-edge yeast three-hybrid (Y3H) platforms for studying plant protein-RNA interactions. Our Y3H technology spans various research areas such as proteomics, genomics, functional genomics, and drug discovery, and helps clients understand the complex biological processes of plants. If you are interested in our services, please contact us for more details.

Reference

  1. Wurster, S. E., & Maher, L. J., 3rd (2010). Selections that optimize RNA display in the yeast three-hybrid system. RNA (New York, N.Y.). 16(2), 253-258.

For research use only, not for clinical use.