Question

What assay uses the fact that activators can be separated into DNA binding and activating domains? What information do you gain from this assay? Describe briefly how to perform this assay.

Answer 1

Two-hybrid screening is the assay based on the fact that activators have separate DNA binding and activation domains.

Two-hybrid screening approaches are high-throughput complementation assays that test for protein–protein or protein–DNA interactions.

The assay is based on the fact that transcription factors have separate domains for DNA binding and activation.The assay is typically performed by introducing proteins of interest pairwise into yeast, with each protein fused to a transcription factor that has been split into two complementary fragments.The two domains can be separated from each other in which case the protein loses its activity.Plasmids are engineered to produce a protein product in which the DNA-binding domain (BD) fragment is fused onto a protein (bait protein) while another plasmid is engineered to produce a protein product in which the activation domain (AD) fragment is fused onto another protein (prey protein).When brought into close proximity to one another through interaction between the bait and prey proteins, the binding and activation domains of the transcription factor function to activate transcription of a reporter gene. If the reporter gene is expressed it means that there has been interaction of the bait protein with the prey protein.