Identification of Ecdysone Receptor Coregulators by Yeast Two-Hybrid Assay
Disciplines
Biochemistry | Molecular Biology
Abstract (300 words maximum)
Ecdysone, a steroid hormone, has a crucial role in the development of insects and other arthropods. The ecdysone receptor is a heterodimer of EcR and ultraspiracle protein (USP). The ligand binding domain (LBD) located on the EcR subunit of the ecdysone receptor is the region that binds ecdysone and other proteins to regulate gene expression. Though the ecdysone receptor has been modeled in detail, the identity of the proteins that bind LBD is understudied and is the purpose of this research. A yeast two-hybrid (Y2H) assay is used to identify protein interactions with the LBD of the ecdysone receptor. Y2H assay uses the Gal4 transcription factor domains called DNA binding domain (BD) and the activation domain (AD). The LBD of the ecdysone receptor is fused with the BD of Gal4, acting as a ‘bait’ and the AD of Gal4 is fused with proteins from a drosophila cDNA library acting as the ‘prey’. Gal4 is reconstituted when the two halves are brought into proximity by the interaction of bait and prey proteins enabling the activation of reporter genes, most prominent being the activation of alpha-galactosidase which in the presence of X-alpha gal gives blue-colored colonies indicative of protein-protein interactions. Control experiments were conducted to ensure assay validity. Autoactivation testing was performed to confirm that the bait construct does not independently activate the reporter gene in the absence of prey. For positive control, a known interacting protein pair (p53 and T-antigen) was used to confirm that the Y2H system was functioning correctly. Negative control was performed by two non-interacting proteins (T-antigen and laminin) to verify that no non-specific interactions occurred. With controls successfully completed, the Y2H screen was conducted to identify proteins interacting with the ecdysone receptor. Positives will be further analyzed to identify novel proteins interacting with the ecdysone receptor.
Academic department under which the project should be listed
CSM - Chemistry and Biochemistry
Primary Investigator (PI) Name
Dr. Rajnish Singh
Identification of Ecdysone Receptor Coregulators by Yeast Two-Hybrid Assay
Ecdysone, a steroid hormone, has a crucial role in the development of insects and other arthropods. The ecdysone receptor is a heterodimer of EcR and ultraspiracle protein (USP). The ligand binding domain (LBD) located on the EcR subunit of the ecdysone receptor is the region that binds ecdysone and other proteins to regulate gene expression. Though the ecdysone receptor has been modeled in detail, the identity of the proteins that bind LBD is understudied and is the purpose of this research. A yeast two-hybrid (Y2H) assay is used to identify protein interactions with the LBD of the ecdysone receptor. Y2H assay uses the Gal4 transcription factor domains called DNA binding domain (BD) and the activation domain (AD). The LBD of the ecdysone receptor is fused with the BD of Gal4, acting as a ‘bait’ and the AD of Gal4 is fused with proteins from a drosophila cDNA library acting as the ‘prey’. Gal4 is reconstituted when the two halves are brought into proximity by the interaction of bait and prey proteins enabling the activation of reporter genes, most prominent being the activation of alpha-galactosidase which in the presence of X-alpha gal gives blue-colored colonies indicative of protein-protein interactions. Control experiments were conducted to ensure assay validity. Autoactivation testing was performed to confirm that the bait construct does not independently activate the reporter gene in the absence of prey. For positive control, a known interacting protein pair (p53 and T-antigen) was used to confirm that the Y2H system was functioning correctly. Negative control was performed by two non-interacting proteins (T-antigen and laminin) to verify that no non-specific interactions occurred. With controls successfully completed, the Y2H screen was conducted to identify proteins interacting with the ecdysone receptor. Positives will be further analyzed to identify novel proteins interacting with the ecdysone receptor.