Question
Describe how epitope tagging can be used to purify polymerase II from yeast in one step.
Step 1
Common tags include FLAG, HA, or His tags. The tag should be small, not interfere with the enzyme's function, and have a high affinity for a specific antibody or binding partner. Show more…
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Explain how the subunits of RNA polymerase II of yeast were identified using epitope tagging.
The modular nature of eukaryotic activator proteins gave scientists an idea for a way to find proteins that interact with any particular protein of interest. The idea is to use the protein-protein interaction to bring together a DNA-binding region with an activation region, creating an artificial activator that consists of two polypeptides held together noncovalently by the interaction.The method is called the yeast two-hybrid system, and it has three components. First, the yeast contains a reporter gene construct in which $U A S_{G}$ (an enhancerlike sequence that binds the activator Gal4 as described in Problem 8 drives the expression of an $E$ coli lacZ reporter (encoding the enzyme B-galactosidase) from a yeast promoter. Second, the yeast also expresses a fusion protein in which the DNA-binding domain of Gal4 is fused to the protein of interest; this fusion protein is called the bait. The third component is a cDNA library made in plasmids, where each cDNA is fused in frame to the activation domain of Gal4, and these can be expressed in yeast cells as prey fusion proteins. How could you use a yeast strain containing the first two components, along with the plasmid cDNA expression library described, to identify prey proteins that bind to the bait protein? How is this procedure relevant to the goal of finding proteins that might interact with each other in the cell?
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