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 ID: 374367
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Slide1
Controlling protein overexpression from yeast shuttle vectors
GAL1
promoter is induced by galactoseSlide2
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?Slide3
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
promoterSlide4
master plate
Y
C-
ura
orientation marker
Y
C-
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 sourcesSlide5
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?Slide6
Yeast must activate alternative pathways when galactose replaces glucose
Transcription patterns change when galactose replaces glucose
Glucose is the preferred carbon source for yeast
adaptationSlide7
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 processesSlide8
Few substrates for
glycolysis
Little energy produced
Cells need to adjust their transcriptional program when glucose is not available
Galactose
= Slide9
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
PSlide10
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?Slide11
P
romoters of the
GAL7
,
GAL10
and
GAL1
genes contain multiple binding sites for the Gal4p transcriptional activatorSlide12
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 metabolismSlide13
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
)CCGSlide14
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 galactoseSlide15
Inhibitory proteins
Glucose
represses transcription
Transcription
Galactose
activates transcription ~1000-fold
some transcription
Raffinose
relieves glucose repression