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Gene regulation
Positive vs. negative control
Positive control: A protein binds to DNA and transcription increases
mRNA
5’
3’
CAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACATTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCT
GTTGCGTTAATTACACTCAATCGAGTGAGTAATCCGTGGGGTCCGAAATGTAAATACGAAGGCCGAGCATACAACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGA
|
|
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-35
-10
+1
Activator
Negative control: A protein binds to DNA and transcription decreases
5’
3’
X
mRNA
CAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACATTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCT
GTTGCGTTAATTACACTCAATCGAGTGAGTAATCCGTGGGGTCCGAAATGTAAATACGAAGGCCGAGCATACAACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGA
|
|
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-35
-10
+1
Repressor
The lac operon
What is an operon?
A collection of genes that are transcribed together and often have a
related function (in this case lactose metabolism).
Lactose
The lac operon: negative regulation
Promoter
DNA covered by RNA polymerase
5’
3’
LacI
X
mRNA
CAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACATTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCT
GTTGCGTTAATTACACTCAATCGAGTGAGTAATCCGTGGGGTCCGAAATGTAAATACGAAGGCCGAGCATACAACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGA
|
|
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-35
-10
+1
DNA covered by repressor
Operator
Without lactose, LacI binds to the operator, which prevents RNA polymerase
from binding to the promoter
The lac operon: negative regulation
Promoter
DNA covered by RNA polymerase
5’
3’
LacI
X
mRNA
CAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACATTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCT
GTTGCGTTAATTACACTCAATCGAGTGAGTAATCCGTGGGGTCCGAAATGTAAATACGAAGGCCGAGCATACAACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGA
|
|
|
-35
-10
+1
DNA covered by repressor
LacI
Operator
+
Lactose
If lactose is added, it binds LacI and stabilizes the LacI conformation that doesn’t
bind to the operator.
The lac operon: negative regulation
Promoter
DNA covered by RNA polymerase
mRNA
5’
3’
CAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACATTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCT
GTTGCGTTAATTACACTCAATCGAGTGAGTAATCCGTGGGGTCCGAAATGTAAATACGAAGGCCGAGCATACAACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGA
|
|
|
-35
-10
+1
DNA covered by repressor
LacI
Operator
When LacI is not bound to the operator, the promoter binding site is not occluded.
Keq of DNA binding
The graph to the right shows two
curves that represent binding of LacI
to the operator with and without
lactose.
100
% DNA
bound
Which curve has a higher Keq for
binding to DNA?
50
0
[LacI]
The red curve
Operator DNA + LacI
[O]
[O − R]
Keq =
O [R]
=
1
R
[R]
Keq
LacI/Operator Complex
[O-R]
Keq for binding is inversely related to the concentration
of LacI at which 50% of the DNA is bound
Keq of DNA binding
The graph to the right shows two
curves that represent binding of LacI
to the operator with and without
lactose.
Which curve represents binding of
LacI without lactose?
Higher Keq
100
% DNA
bound
Lower Keq
50
0
[LacI]
The red curve – LacI has a higher affinity for DNA when
lactose is absent.
The lac operon: positive regulation
Dissociation of LacI from the operator is not sufficient to activate transcription of
the lac operon, it also requires positive regulation.
CAP Binding Site
Promoter
mRNA
5’
3’
CAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACATTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCT
GTTGCGTTAATTACACTCAATCGAGTGAGTAATCCGTGGGGTCCGAAATGTAAATACGAAGGCCGAGCATACAACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGA
|
|
|
-35
-10
+1
Why is positive regulation necessary?
The lac promoter -10 and -35 regions poorly
match the consensus sequence, and RNA
polymerase binds poorly to them.
Operator
The lac operon: positive regulation
CAP, the activator, can only bind to the DNA when cAMP levels are high. This only
occurs when glucose levels are low.
CAP Binding Site
5’
3’
Promoter
mRNA
CAP
CAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACATTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCT
GTTGCGTTAATTACACTCAATCGAGTGAGTAATCCGTGGGGTCCGAAATGTAAATACGAAGGCCGAGCATACAACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGA
|
|
|
-35
-10
+1
Operator
CAP
+
cAMP
The lac operon: positive regulation
CAP, the activator, can only bind to the DNA when cAMP levels are high. cAMP
levels are high when glucose levels are low.
CAP Binding Site
5’
3’
Promoter
mRNA
CAP
CAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACATTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCT
GTTGCGTTAATTACACTCAATCGAGTGAGTAATCCGTGGGGTCCGAAATGTAAATACGAAGGCCGAGCATACAACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGA
|
|
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-35
-10
+1
Operator
CAP binding to the CAP binding site helps recruit RNA polymerase, allowing it to
bind to the -35 and -10 sites that poorly match the consensus sequence.
Electrophoretic mobility shift assays (EMSA)
The affinity of a certain repressor for binding
to its operator site is affected by the
presence of molecule “A.” Given these EMSA
data, would the addition of molecule A
increase or decrease transcription of the
gene controlled by this repressor?
Without molecule A:
Keq = 109
With molecule A:
Keq = 1011
The affinity of the repressor for DNA is
higher for higher when molecule A is
present. The addition of molecule A would
increase repressor binding to DNA,
decreasing transcription.
[repressor] 10-12 10-11 10-10 10-9 10-8
[DNA]
Constant
without
molecule A
[repressor] 10-12 10-11 10-10 10-9 10-8
[DNA]
with
molecule A
Constant
The lac operon
Glucose
-10 Region
CAP-Binding Site
LacZ
RNA Polymerase
Operon
LacI
cAMP
-35 Region
Consensus Sequence
Lactose
Repressor
CAP
Activator
Promoter
Operator
The lac operon: a concept map
prevents LacI binding
to the operator
Lactose
encodes the genes
needed to use
Is a
Repressor
LacI
binds to a site in
the DNA called the
lac Operon
negatively
regulates
Operator
positively
regulates
Binds to a site in
the DNA called the
Glucose
Promoter
comprise
CAP
CAP
Binding Site
encodes
LacZ
DNA that promotes
transcription of
is an
Activator
used to metabolize
binds
-10 Region
-35 Region
RNA Polymerase
binds
binds
Mediates CAP
binding to DNA
cAMP
cAMP is produced when
glucose is depleted
Consensus Sequence
Ideal -10/-35 sequences to which
RNA polymerase binds best