ECDYSONE RECEPTOR PROTEIN INTERACTIONS PROFILE
Disciplines
Biochemistry | Molecular Biology
Abstract (300 words maximum)
Ecdysone is a steroid hormone critical for developmental processes in insects. Ecdysone binds to the ligand-binding domain (LBD) of the ecdysone receptor to begin metamorphosis. The LBD interacts with ultra-spiracle protein (USP), and an unknown number of other proteins to modulate gene expression. The identities of these proteins and understanding of their interactions with the LBD and USP, remains limited. The advanced biochemistry CURE lab will use a yeast 2 hybrid assay which takes advantage of the DNA binding and activation domains of the GAL- 4 transcription factor. A fusion protein is composed of the binding domain (BD) and the LBD encoded on the pGBKT7 (bait plasmid, BD-LBD). The activation domain (AD) is fused to proteins from a cDNA drosophila library encoded on pGADT7 (prey plasmids). An interaction between a prey protein and bait protein will reconstitute the GAL4 transcription factor and induce expression of reporter genes resulting in blue colonies. To ensure the validity of the yeast two hybrid assay, a series of controls were run. For the positive control, 2 known interacting proteins: T-antigen (TAg) and p53 were used and for the negative control non-interacting proteins TAg and Laminin (LAM) were used. A yeast two hybrid assay using AD-TAg and BD-p53 produced blue colonies while no blue colonies were seen when AD-TAg and BD-LAM were used. We also did the yeast two hybrid assay with only the bait plasmid- BD-LBD and no blue colonies were seen indicating that the bait plasmid does not autoactivate reporter genes. After successful completion of control experiments, a library screen of BD-LBD interacting proteins will be carried and potential interactors identified. This research provides valuable insights with potential applications to the understanding of nuclear receptors.
Academic department under which the project should be listed
CSM - Chemistry and Biochemistry
Primary Investigator (PI) Name
Dr. Rajnish Singh
ECDYSONE RECEPTOR PROTEIN INTERACTIONS PROFILE
Ecdysone is a steroid hormone critical for developmental processes in insects. Ecdysone binds to the ligand-binding domain (LBD) of the ecdysone receptor to begin metamorphosis. The LBD interacts with ultra-spiracle protein (USP), and an unknown number of other proteins to modulate gene expression. The identities of these proteins and understanding of their interactions with the LBD and USP, remains limited. The advanced biochemistry CURE lab will use a yeast 2 hybrid assay which takes advantage of the DNA binding and activation domains of the GAL- 4 transcription factor. A fusion protein is composed of the binding domain (BD) and the LBD encoded on the pGBKT7 (bait plasmid, BD-LBD). The activation domain (AD) is fused to proteins from a cDNA drosophila library encoded on pGADT7 (prey plasmids). An interaction between a prey protein and bait protein will reconstitute the GAL4 transcription factor and induce expression of reporter genes resulting in blue colonies. To ensure the validity of the yeast two hybrid assay, a series of controls were run. For the positive control, 2 known interacting proteins: T-antigen (TAg) and p53 were used and for the negative control non-interacting proteins TAg and Laminin (LAM) were used. A yeast two hybrid assay using AD-TAg and BD-p53 produced blue colonies while no blue colonies were seen when AD-TAg and BD-LAM were used. We also did the yeast two hybrid assay with only the bait plasmid- BD-LBD and no blue colonies were seen indicating that the bait plasmid does not autoactivate reporter genes. After successful completion of control experiments, a library screen of BD-LBD interacting proteins will be carried and potential interactors identified. This research provides valuable insights with potential applications to the understanding of nuclear receptors.