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?
Expression of plasmid-encoded MET genes is regulated by the inducible
GAL1 promoter
S. pombe Met
or lacZ
S. cerevisiae MET
GAL1 promoter
GAL1 promoter
pBG1805
pYES2.1
URA3
URA3
promoter
URA3
promoter
URA3
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 to using galactose as a carbon source?
How is the GAL1 promoter regulated?
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
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
for glycolysis
Little energy produced
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
Glycolysis
P
ENERGY
Glucose =
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?
Promoters of the GAL7, GAL10 and GAL1 genes contain
multiple binding sites for the Gal4p transcriptional activator
Gal4p 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 17bp sequence in promoters
of multiple genes
UAS=upstream activating sequence
MUCH larger transactivation domain is not included in this structure!
GAL1 promoter is subject to both positive and negative regulation
Positive and negative regulatory proteins bind to cis-elements in the
GAL1 promoter
UAS
CGG(N11)CCG
Gal4p binds upstream
activating sequence (UAS)
MET coding sequence
Repressor proteins
bind here when
glucose is available
Galactose relieves Gal4p repression by a complex mechanism
In the absence of galactose,
Gal80p inhibits Gal4p
Glucose
Gal80p not longer binds Gal4p
in presence of galactose
Galactose
Gal80p protein
Gal4p dimer
Activated Gal4p recruits
transcriptional machinery
Glucose represses transcription
Inhibitory proteins
Raffinose relieves glucose repression
some transcription
Galactose activates transcription ~1000-fold
Transcription