Lecture 3
The regulation of gene expression
in eukaryotes. S. cerevisiae Gal4 as
a guide for universal principles of
transcriptional regulation
Tuesday, 6 November 2012
•The Lac operon decides to use or not to use lactose
•The λ phage deciding whether to sail or to jump in an E. coli
cell
•Galactose utilization in yeast
•Making stripes in the Drosophila embryo
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•Eukaryotes need to cater for the same as
bacteria but also sexual reproduction,
more complex and social environments
•Eukaryotes have a more complex
structure more genes, MORE
COMPUTATIONAL POWER
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Saccharomyces cerevisiae
http://es.wikipedia.org/wiki/Archivo:Budding_yeast_Lifecycle.png
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Glucose
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Galactose
Saccharomyces cerevisiae Galactose utilization
Gal1
Gal2
Gal5
Gal7
Gal10
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There are genes that code for enzymes and genes that code for......something else.
Some transcription factors but also other proteins
Gal10
Gal7
Gal4
Gal2
Gal80
Gal3
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Gal1
Gal11
Gal4 encodes a DNA binding protein
Gal10
Gal7
Gal4
Gal2
Gal80
Gal3
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Gal1
Gal11
Reporters facilitates the analysis of gene regulation
TATA
GAL1
ß-galactosidase
Green Fluorescent Protein
GFP
Deletion analysis uncovers a 400 bp region which is sufficient for GAL1
expression, and within it a small region that is essential
GAL1
Tuesday, 6 November 2012
Reporter
+
+
-
Within the 400bp required for expression, 275bp from the transcription start site
there are four binding sites for Gal4 (UASGal4 for Upstream Activating Sequences
for Gal4) which promote GAL1 expression
@ 400bp
275bp
UAS GAL4
Mig1
GAL1
....there is also a binding site for a repressor, Mig1, which represses GAL1
expression in the presence of Glucose.
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Every gene encoding enzymes has a Gal4 binding site
Gal10
Gal7
Gal4
Gal2
Gal80
Gal3
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Gal1
Gal11
Gal1
Gal4
Gal80
Gal3
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Gal11
Mig1 is a repressor which ensures that GAL genes are only used when there is no Glucose
around. It works in a different manner from bacterial repressors as it acts by recruiting a number
of proteins which make the promoter inaccesible to RNA polymerase
From Ptashne and Gann, Genes and signals
Tuesday, 6 November 2012
From Ptashne and Gann, Genes and signals
Tuesday, 6 November 2012
Gal4
Gal80
Gal3
Gal11
Gal3 shuttles between the cytosol and the nucleus and, in
the presence of Galactose, binds Gal80 releasing the
activity of Gal4 and providing a rapid induction of
transcriptional activity
No galactose
Galactose
Gal80
Gal4
Mig1
Mig1
GAL1
GAL1
Gal3
OFF
Gal3
Mig1
GAL1
Mig1
GAL1
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ON
Gal4
Gal11 is a component of the MEDIATOR complex which,
through an interaction with Gal4, allows the link between
Gal4 and the basal transcriptional machinery
Gal80
Gal3
Gal11
Gal80
GAL1
Galactose
Gal11
Gal4
MEDIATOR
Mig1
GAL1
RNA polII
GAL1
Tuesday, 6 November 2012
Gal4 is a transcriptional regulator
λ repressor (cI)
!
From Ptashne and Gann, Genes and signals
Tuesday, 6 November 2012
λ repressor (cI)
!
From Ptashne and Gann, Genes and signals
Tuesday, 6 November 2012
!
The Gal4 protein is structurally and functionally MODULAR
From Ptashne and Gann, Genes and signals
Tuesday, 6 November 2012
The Gal4 protein is structurally and functionally MODULAR
From Ptashne and Gann, Genes and signals
LexA is a bacterial transcriptional regulator with a high affinity DNA binding site.
Of course one has to provide a LexA binding site in yeast but this is no problem.
Tuesday, 6 November 2012
The Gal4 protein is structurally and functionally MODULAR
LexA binding site
From Ptashne and Gann, Genes and signals
LexA is a bacterial transcriptional regulator with a high affinity DNA binding site.
Of course one has to provide a LexA binding site in yeast but this is no problem.
Tuesday, 6 November 2012
How specific is the AD of Gal4?
From Ptashne and Gann, Genes and signals
Tuesday, 6 November 2012
The modular nature of transcriptional regulators
DNA binding domain
Gal4, LexA, cI....
Activation domain
VP16, Gal4,.......
Repression domain
enR, Mig1,.....
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Transcription factors are modular
DNA binding
Trans Activation
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Yeast Gal4 works in Drosophila
cell type or
tissue specific enhancer
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lacZ
Fluorescent proteins
cell type or
tissue specific enhancer
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....and in fish
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Summary
•The structure of an eukaryotic cells mirrors (suits) its
computational capacity.
•Regulated recruitment and cooperativity are central elements
of the transcriptional process that promotes functionality with a
‘small’ number of transcription factors.
•Transcription factors are modular (DNA binding + Activator
or Repressor domains).
•The interactions between transcription factors and the basal
transcriptional machinery are Universal.
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Drosophila development
is about making
segments, making stripes
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In bicoid (bcd) mutants, all anterior structures are missing
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bicoid encodes a transcription factor that distributes itself in a gradient across the embryo (it can
do this because the embryo is a syncitium at this early stage)
Bicoid protein
Bcd
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The position of pattern landmarks is determined by the local
concentration of Bicoid
http://scienceblogs.com/pharyngula/
Tuesday, 6 November 2012
Bicoid encodes a transcription factor and the hunchback (hb)
gene is a target of Bicoid which responds at a certain
concentration
Bicoid (bcd)
AD
HD
Bcd
hunchback
(hb)
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No Bicoid (bcd)
The combination of Bicoid binding sites with different affinities in the
promoter of hunchback, determines the pattern of response; the domain
of expression of the hunchback gene
Bicoid gradient
hunchback or synthetic
binding sites
High affinity
Tuesday, 6 November 2012
Low affinity
reporter expression in embryo
The combination of Bicoid binding sites with different affinities in the
promoter of hunchback, determines the pattern of response; the domain
of expression of the hunchback gene
Bicoid gradient
hunchback or synthetic
binding sites
High affinity
Low affinity
Synthetic High affinity
Synthetic Low affinity
Tuesday, 6 November 2012
reporter expression in embryo
Bicoid has a DNA binding domain, homeodomain (HD) and
an activation domain (AD) which can be substituted by the
yeast Gal4 AD.
Bicoid (bcd)
AD
HD
bcd
hb bs
GAL1 lacZ
o
o
Gal4 AD
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A cascade of transcription factors
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Transcription factor gradients activate even skipped expression.
Borok M J et al. Development 2010;137:5-13
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Bcd
Hb
eve 2
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Gt
Kr
Bcd
Bcd
Hb
Gt
Kr
Hb
eve 2
eve 2
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In summary
•The spatial localization of domains of gene expression
linked to pattern formation relies on the deployment of
transcription factors in specific domains.
•The deployment is mediated by transcription factors
or combinations of transcription factors (activators and
repressors).
•The process relies on a combination of regulated
recruitment and differential affinities.
•The programmes are time chains of transcriptional
regulation.
•The transcriptional mechanism is universal.
Tuesday, 6 November 2012
Positive autoregulatory loop
Bistable switch
Feedforward loop
Bcd
lacZYA
I
p
int
L
N
p
cI
cro
RE
cII
O P
Hb
p
R
Y
lactose
Gt
eve 2
Bcd
lac Operon
cI
cro
Hb
Lysogeny
Lysis
eve 2
Tuesday, 6 November 2012
Kr