Question

Previous experiments with the Cdc16 and Cdc23 mutants suggested that the encoded proteins work together to execute common functions. Investigators hypothesized that they exist in a protein complex. You decided to use yeast two hybrid assay to show that these two proteins interact. Explain in your own words how you would do this.

Answer 1

In a yeast two hybrid system, to show that the cdc16 and cdc23 interact with each other forming a complex, one must first clone the cdc16 into the bait protein which will be a complex of the DNA binding domain of the yeast and the cdc16 protein then the another clone must be the cdc23 cloned into the prey protein which will be a complex of the activation domain of the yeast and the cdc23 protein. These two clones would be expressed inside the yeast cell together and they would produce the proteins as above i.e. one bait+cdc16 and one prey+cdc23. If the two proteins are interacting then the transcription of the reporter gene in the yeast would be activated and the yeast colonies would be different colored. Thus indicating that the two proteins are interacting.

As controls, two more plates should be taken and yeast transformed with only bait+cdc16 plasmid should be grown and on another plate yeast transformed with only prey+cdc23 plasmid should be grown. These will be colorless colonies.

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