Question

GAL3 protein binds to GAL80 and frees up GAL4 to bind to UAS and activate transcription of genes involved in galactose metabolism (see text). Assuming that you did not have prior knowledge of GAL3 protein but knew about GAL4 and GAL80, what experiments (genetic screens or selection experiments) would you perform to identify the GAL3 gene and additional genes involved in the regulation of GAL4?

Answer 1

To identify transcriptional regulators of GAL4, we can perform the yeast-1-hybrid assay.
The upstream region of the GAL4 coding sequence cloned along with a reporter gene such as GUS.
Yeast transcription factor clones are mated with this strain. Colonies that produce blue color indicate that a given transcription factor binds to the upstream region.

To identify post-translational regulators or interacting factors at the protein level, we can perform several experiments.
1. Yeast-2-hybrid assay: Gal4 is used as a bait to identify unknown interacting proteins.
2. Coimmunoprecipitation: Gal4 is pulled down with a specific antibody for the cell lysed. Then, the lysate is probed with specific antibodies against other proteins. We can also perform Mass-spectrometry on the pulled-down sample to identify the interacting factors.
3. We can also perform phage display