B6. Gall gene encodes enzyme galactokinase which phosphorylates sugar galactose

Question

B6. Gall gene encodes enzyme galactokinase which phosphorylates sugar galactose for catabolism (utilized for energy). Gall gene is activated when sugar galactose is present and sugar glucose is absent in the medium. Gall gene is not activated by galactose when glucose is present. This is because transcription factor MIG1 binds to MIG1 site in the Gall promoter and recruits TUPI to repress the Gall gene in yeast when glucose is present in the medium. (1) How will you determine that MIG1 binds to MIGl site only w present in the medium and not present when galactose (no glucose) in the medium. (2) How will you establish that MIG1 transcription factor forms a complex with TUP1? hen glucose is

Explanation / Answer

1. MIG1 is tagged with any fluorescent protein (eg. Green fluorescent protein) and examined its localization in presence and absence of glucose. In presence of glucose, MIG1 goes into the nucleus and binds to the DNA and in glucose absence its exported outside nucleus.

2. One way to do is use a TUP1 knockdown S.cerevisiae and check for expression level of genes corresponding to galactose metabolism. In mutant form, there is more expression of genes compared to normal organism. It partially indicates that TUP1 binds to MIG1 in shut down of galactose genes.

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