You are a researcher who has just discovered a new protein in a fungus. To help
ID: 90918 • Letter: Y
Question
You are a researcher who has just discovered a new protein in a fungus. To help determine the protein's function, you want to identify the other proteins in the fungal cell with which your protein interacts. How do you design a yeast two-hybrid experiment to address this problem?
a) Express the protein in bacterial strain 1 as a fusion protein with the DNA-binding domain of Gal4p. Using bacterial strain 2, make a library representing all fungal proteins expressed as fusion proteins with the Gal4p activation domain. Encourage conjugation between strain 1 and strain 2, and look for colonies that express a reporter gene. These colonies will generally arise from cells containing fusion protein that interacts with your target protein. b) Express the protein in yeast strain 1 as a fusion protein with the ribosomal-binding domain of Gal4p. Using yeast strain 2, make a library representing all fungal proteins expressed as fusion proteins with the Gal4p activation domain. Mate strain 1 with the strain 2 library, and look for colonies that express a reporter gene. These colonies will generally arise from mated cells containing fusion protein that interacts with your target protein. c) Express the protein in yeast strain 1 as a fusion protein with the DNA-binding domain of Gal4p. Using yeast strain 2, make a library representing all fungal proteins expressed as fusion proteins with the Gal4p activation domain. Mate strain 1 with the strain 2 library, and look for colonies that express a reporter gene. These colonies will generally arise from mated cells containing fusion protein that interacts with your target protein. d) Express the protein in yeast strain 1 as a fusion protein with the DNA-binding domain of Gal4p. Using yeast strain 2, make a library representing all fungal proteins expressed as fusion proteins with the Gal4p activation domain. Mate strain 1 with the strain 2 library, and look for colonies that express the target gene, which creates either a color or another selective advantage. These colonies will generally arise from mated cells containing fusion protein that interacts with your target protein.Explanation / Answer
Answer) The correct option is c) Express the protein in yeast strain 1 as a fusion protein with the DNA-binding domain of Gal4p. Using yeast strain 2, make a library representing all fungal proteins expressed as fusion proteins with the Gal4p activation domain. Mate strain 1 with the strain 2 library, and look for colonies that express a reporter gene. These colonies will generally arise from mated cells containing fusion protein that interacts with your target protein.
Yeast-two hybrid system is based on the fascinitaing properties of the GAL4 protein which is known to be found in the baker's yeast Saccharomycese cerevisiae. This is used as a model for studying the transcriptional activation of proteins. The transcriptional activity of Gal4p depends on the DNA binding activity at its amino terminus and an activation domain present at its carboxy terminus. The mating of yeasts reconstitute a functional transcription factor that is able to induce reporter gene expression.
Related Questions
Navigate
Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.