1. The yeast two-hybrid system is a powerful molecular genetic method to identif

Question

1.The yeast two-hybrid system is a powerful molecular genetic method to identify a protein(s) that interacts with a known protein or protein domain. You have isolated the glucocorticoid receptor (GR) and have evidence that is a modular protein containing an activation domain, a DNA binding domain and a second ligand binding-activation domain. Further analysis reveals that in the pituitary cells the GR is anchored in the cytoplasm in the absence of its hormone ligand. This result lead you to speculate that GR binds to an inhibitory protein in the cytoplasm of the pituitary cells. (i) Describe how a yeast-two hybrid analysis system could help you to identify the protein with which GR interacts. ii) How would you specifically identify the domain in the GR that binds the inhibitor? (20%)

Explanation / Answer

i) The Glucocorticoid receptor (GR) is a zinc finger doamin. The zinc finger domain is generally located between 23 and 28 amino acids long and is stabilized by coordinating zinc ions with regularly spaced zinc-coordinating residues (either histidines or cysteines). The most common class of zinc finger (Cys2His2) coordinates a single zinc ion and consists of a recognition helix and a 2-strand beta-sheet. In transcription factors these domains are often found in arrays and adjacent fingers are spaced at 3 basepair intervals when bound to DNA. In the GR bound to DNA is a zinc finger model in which the zinc atoms coordinating with cysteine side chains. In the yeast two hybrid system, the DNA binding domain is inactivated to study the transcription regulation in one of the yeast cell (mutant) while the other being wild type. In another yeast cell, the ligand activating domain is inactivated to study the expression of the gene.

ii) Normally, GR is a zinc finger domain, in which the GR binds to DNA through zinc atoms and cysteine side chains. If the GR binds to inhibitory molecule it no longer binds to zinc atoms and cysteine sidechain molecules are not exposed for regulation. This leads to alternative confirmation of GR and change in its shape is observed. By this it can be known that GR is bound to an inhibitor molecule.

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