Dna

Binding

LacZ

P Gal4 Binding site

LacZ

LacZ P Gal4 Binding site

Figure 8 The yeast-two hybrid system. (A) The ''wild-type'' GAL4 transcription factor has two domains, the activating and the DNA-binding domains. (B) Two interacting proteins X and Y that are chimeras with the DNA-binding domain of GAL4 and the activating domain of GAL4-respectively. When the two proteins interact, the activating domain and the DNA-binding domain are brought together to function as a transcription factor.

LacZ P Gal4 Binding site

Figure 8 The yeast-two hybrid system. (A) The ''wild-type'' GAL4 transcription factor has two domains, the activating and the DNA-binding domains. (B) Two interacting proteins X and Y that are chimeras with the DNA-binding domain of GAL4 and the activating domain of GAL4-respectively. When the two proteins interact, the activating domain and the DNA-binding domain are brought together to function as a transcription factor.

provides a powerful selection procedure. This FOA system is used in the ''reverse two-hybrid'' system, providing a selective growth advantage and a more powerful system for screening. In the reverse two-hybrid system, the interacting protein is expressed inducibly, and only when the interacting proteins are blocked do the cells survive. GAL4 and LexA transcription factors are most often used in the yeast two-hybrid system. In two-hybrid screens, it is useful to have two separate reporter constructs to help in sorting ''hits.'' Reporters such as LEU2 and LacZ can be expressed in the same cell.

The yeast 2-hybrid system has been used to develop screens for ligand-receptor interactions, including peptide hormone receptors and the tyrosine kinase receptors [45-47]. Specific and reversible ligand-receptor interactions between growth hormone and growth hormone receptor, VEGF and KDR, can be studied using the yeast two-hybrid system. Ligand-dependent receptor dimerization can also be studied using three expression plasmids in which the receptor is expressed as a fusion protein with both the DNA binding domain as well as the activation domain. The ligand is expressed from a third plasmid. When the ligand binds the two receptors, the DNA-binding domain and activating domains are pulled together and Gal4 is activated (Fig. 9).

Figure 9 The yeast three-hybrid system. Yeast systems to identify ligand-receptor interactions are shown. In the example shown, (A) growth hormone is expressed as a chimera with the DNA-binding domain of GAL4 under the control of a regulatable promoter (copper). Growth hormone receptor is expressed as a chimera of the activating domain of GAL4. When induced, the activating and binding domains are brought together by the interaction of growth hormone with its receptor. This is a two-hybrid system. (B) In contrast, in the three-hybrid system, both the DNA-binding and activation domains are expressed as chimeras with growth hormone receptor. Growth hormone is expressed from a third plasmid, under regulation of the inducible copper promoter. When uninduced, the activating and binding domains are not brought together. When induced, the excess of growth hormone binds the growth hormone receptor, inducing dimerization and functional activation of GAL4, resulting in the expression of HIS3 reporter.

Figure 9 The yeast three-hybrid system. Yeast systems to identify ligand-receptor interactions are shown. In the example shown, (A) growth hormone is expressed as a chimera with the DNA-binding domain of GAL4 under the control of a regulatable promoter (copper). Growth hormone receptor is expressed as a chimera of the activating domain of GAL4. When induced, the activating and binding domains are brought together by the interaction of growth hormone with its receptor. This is a two-hybrid system. (B) In contrast, in the three-hybrid system, both the DNA-binding and activation domains are expressed as chimeras with growth hormone receptor. Growth hormone is expressed from a third plasmid, under regulation of the inducible copper promoter. When uninduced, the activating and binding domains are not brought together. When induced, the excess of growth hormone binds the growth hormone receptor, inducing dimerization and functional activation of GAL4, resulting in the expression of HIS3 reporter.

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