Controlling protein overexpression from yeast shuttle vectors GAL

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Controlling protein overexpression from yeast shuttle vectors GAL 1 promoter is induced by galactose

Controlling protein overexpression from yeast shuttle vectors GAL 1 promoter is induced by galactose

How is replica plating used to analyze MET gene complementation? How do cells adapt

How is replica plating used to analyze MET gene complementation? How do cells adapt to using galactose as a carbon source? How is the GAL 1 promoter regulated?

Expression of plasmid-encoded MET genes is regulated by the inducible GAL 1 promoter S.

Expression of plasmid-encoded MET genes is regulated by the inducible GAL 1 promoter S. pombe Met or lac. Z S. cerevisiae MET GAL 1 promoter p. BG 1805 p. YES 2. 1 URA 3 promoter URA 3

Transformed cells will be replica plated on media with different carbon sources Step 4

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

How is replica plating used to analyze MET gene complementation? How do cells adapt

How is replica plating used to analyze MET gene complementation? How do cells adapt to using galactose as a carbon source? How is the GAL 1 promoter regulated?

Glucose is the preferred carbon source for yeast adaptation Yeast must activate alternative pathways

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

Glucose is the preferred carbon source for yeast Glycolysis Glucose is transported into the

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

Cells need to adjust their transcriptional program when glucose is not available Few substrates

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

Cells increase the expression of proteins that: transport galactose in the cell (Gal 2

Cells increase the expression of proteins that: transport galactose in the cell (Gal 2 p) convert galactose into glucose-1 -P (Gal 1 p, Gal 7 p and Gal 10 p) P P Glycolysis P ENERGY Glucose = Galactose =

How is replica plating used to analyze MET gene complementation? How do cells adapt

How is replica plating used to analyze MET gene complementation? How do cells adapt to using galactose as a carbon source? How is the GAL 1 promoter regulated?

Promoters of the GAL 7, GAL 10 and GAL 1 genes contain multiple binding

Promoters of the GAL 7, GAL 10 and GAL 1 genes contain multiple binding sites for the Gal 4 p transcriptional activator

Gal 4 p acts as a master transcriptional regulator Multi-domain protein that binds DNA

Gal 4 p acts as a master transcriptional regulator Multi-domain protein that binds DNA and activates transcription of multiple genes involved in galactose metabolism Dimerization domains Hydrophobic residues on one face of each helix bind the two subunits together DNA binding domains Each contains a Zn finger that coordinates two zinc atoms binds 17 bp sequence in promoters of multiple genes UAS=upstream activating sequence MUCH larger transactivation domain is not included in this structure!

GAL 1 promoter is subject to both positive and negative regulation Positive and negative

GAL 1 promoter is subject to both positive and negative regulation Positive and negative regulatory proteins bind to cis-elements in the GAL 1 promoter UAS CGG(N 11)CCG Gal 4 p binds upstream activating sequence (UAS) MET coding sequence Repressor proteins bind here when glucose is available

Galactose relieves Gal 4 p repression by a complex mechanism In the absence of

Galactose relieves Gal 4 p repression by a complex mechanism In the absence of galactose, Gal 80 p inhibits Gal 4 p Glucose Gal 80 p not longer binds Gal 4 p in presence of galactose Gal 80 p protein Gal 4 p dimer Activated Gal 4 p recruits transcriptional machinery

Glucose represses transcription Inhibitory proteins Raffinose relieves glucose repression some transcription Galactose activates transcription

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