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 Metor lacZ

Expression of plasmid-encoded MET genes is regulated by the inducible GAL1 promoter

master plateYC-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 genesUAS=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

UASCGG(N11)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