Controlling protein overexpression from yeast shuttle vectors GAL1 promoter is induced by galactose

How is replica plating used to analyze MET gene complementation? How do cells adapt to using galactose as a carbon source? How is the GAL1 promoter regulated?

pBG1805 pYES2.1 GAL1 promoter URA3 promoter URA3 S. cerevisiae MET GAL1 promoter URA3 promoter URA3 S. pombe Met or lacZ Expression of plasmid-encoded MET genes is regulated by the inducible GAL1 promoter

master plate YC-ura orientation marker YC-ura YC-met (galactose) YC-met (glucose) Step 1 - transfer colonies to sterile velveteen with gentle tapping Step 2 – transfer colonies to various media Step 3 - Incubate plates at 30˚C Step 4 – Score plates for growth Transformed cells will be replica plated on media with different carbon sources

How is replica plating used to analyze MET gene complementation? How do cells adapt to using galactose as a carbon source? How is the GAL1 promoter regulated?

Yeast must activate alternative pathways when galactose replaces glucose Transcription patterns change when galactose replaces glucose Glucose is the preferred carbon source for yeast adaptation

Glucose is the preferred carbon source for yeast Glucose = Glycolysis ENERGY! Glucose is transported into the cell and is used to generate energy through glycolysis and downstream processes

Few substrates for glycolysis Little energy produced Cells need to adjust their transcriptional program when glucose is not available Galactose =

Glycolysis ENERGY Glucose = Galactose = Cells increase the expression of proteins that: transport galactose in the cell (Gal2p) convert galactose into glucose-1-P (Gal1p, Gal7p and Gal10p) P P P

How is replica plating used to analyze MET gene complementation? How do cells adapt to using galactose as a carbon source? How is the GAL1 promoter regulated?

Promoters of the GAL7, GAL10 and GAL1 genes contain multiple binding sites for the Gal4p transcriptional activator

DNA binding domains Each contains a Zn finger that coordinates two zinc atoms binds 17bp sequence in promoters of multiple genes UAS=upstream activating sequence Dimerization domains Hydrophobic residues on one face of each helix bind the two subunits together MUCH larger transactivation domain is not included in this structure! Gal4p acts as a master transcriptional regulator Multi-domain protein that binds DNA and activates transcription of multiple genes involved in galactose metabolism

GAL1 promoter is subject to both positive and negative regulation Positive and negative regulatory proteins bind to cis-elements in the GAL1 promoter Gal4p binds upstream activating sequence (UAS) Repressor proteins bind here when glucose is available MET coding sequence UAS CGG(N 11 )CCG

Gal4p dimer Gal80p protein Galactose relieves Gal4p repression by a complex mechanism Activated Gal4p recruits transcriptional machinery Glucose Galactose In the absence of galactose, Gal80p inhibits Gal4p Gal80p not longer binds Gal4p in presence of galactose

Inhibitory proteins Glucose represses transcription Transcription Galactose activates transcription ~1000-fold some transcription Raffinose relieves glucose repression