Control of gene expression
 Receptors and transcription factors
 Inducible expression of b-galactosidase (lac operon)
 Structure of lac operator and repressor
 Positive and negative regulation
 Steroid hormone control of gene expression
 Interference RNA
Control of gene expression
CELL
DNA
transciption factors
mRNA
receptors
proteins
ENVIRONMENT
Receptors are proteins
which trigger
intracellular chemical
signaling in response to
an external stimulus
Transcription factors
are proteins which
control mRNA
synthesis
b-galactosidase is an inducible enzyme
b-galactosidase allows Escherichia
coli to grow using lactose as its only
carbon source
b-galactosidase
- inducer
temps
+ inducer
An inducer is a molecule which
modulates the level of expression of a
protein
The lac operon : a set of co-regulated genes
E.coli DNA
b-galactosidase (z)
ATG
Mutants
defective
constitutive
non-inducible
STOP
permease (y)
ATG
STOP
transacetylase (a)
ATG
STOP
Complementation
z-, y-, a-
Genomic DNA
i-
z+, y+, a+
F’ episome
i+
z-, y+, a+
i-
b-galactosidase expression
-lactose + lactose
+
+
E. coli i-z+
E. coli i+z+
E.coli i-z+/F’i+z-
Regulation of b-galactosidase expression by the lac
repressor
In the absence of the
inducer
p repressor
p o
b-galactosidase
Binding of the repressor to the
operator prevents b-galatosidase
transcription
In the presence of the
inducer
p repressor
p o
b-galactosidase
The inducer binds to the repressor,
which prevents it from interacting with
the operator
Structure of the lac operator
The lac repressor protects the lac operator
from digestion by pancreatic
desoxyribonuclease
The lac operator has a symmetric
nucleotide sequence
Some repressor structures
phage l repressor bound to its
DNA operator
Trp repressor bound to its
DNA operator
Interaction between transcription factors and DNA
Specific interaction : some amino-acids of the transcription factor
directly bind to the outside of DNA bases
Asparagine
Non-specific interaction :
Some amino acids of the
transcription factors bind to
the DNA polyphosphate
chains
Structure of the lac repressor
Operator binding domain
Inducer binding domain
Dimerization domain
The lac repressor changes its conformation upon inducer
binding
Repressor
structure in the
presence of IPTG
IPTG
operator
Repressor
structure in the
absence of IPTG
Dimerization of the lac repressor increases the
affinity for the operator
Specificity in the operatorrepressor interaction
Affinity: K = 10-13 M
Association rate:
kon = 1010 M-1.s-1
Dissociation rate:
koff = 10-3 s-1
In the absence
of inductor
In the presence
of inductor
Efficient capture mechanism,
beyond simple diffusion
size
tailleoperator
de l' opérateur
30
5

6  6.10
taille
du génome
5.10
genome
size
Dual control of the lac operon
lactose
glucose
Structure of the CAP protein
CAP = catabolic gene activator protein
recognized sequences
glucose
cAMP
CAP
TGTGA
Lac operon
Arabinose operon
tryptophanase
galactokinase
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Non-linear regulations …
AMPc
glucose
CAP
fructose
b-galactosidase
lactose
b-galactosidase
expression
b-galactosidase
lac repressor
allolactose
… may give rise to complex processes
 bi-stability
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 hysteresis
Ozbudak et al. (2004) Nature 427: 737-740
 oscillations
Atkinson et al. (2003) Cell 113: 597-607
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Hydrophilic and hydrophobic inducers
Hydrophilic molecule
Hydrophobic molecule
Second messenger production :
example cAMP
soluble hormones,
neurotransmitters
steroid hormones, vitamine D,
retinoic acid, nitric oxide (NO)
Steroid hormones and other hydrophobic inducers
Metabolism,
inflammation
Reproduction
(estrogen)
Male phenotype (androgen)
Thyroid hormone
Tadpole metamorphosis
Osteogenesis
Limb bud outgrowth
(anterio-posterior axis)
Nuclear hormone receptor activation
Glucocorticoide response element (GRE)
Estrogen response element (ERE)
5’-NAGAACANNNTGTTCTN
NTCTTGTNNNACAAGAN-5’
5’-NAGGTCANNNTGACCTN
NTCCAGTNNNACTGGAN-5’
Estradiol receptor structure
DNA binding domain
estradiol
estrogen binding domain
Controlled gene knock-out by interference RNA (RNAi)
Long double stranded RNA (dsRNA)
( >30 base pairs)
dsRNA-activated
protein kinase
PKR
ATF2
elFa
NF-kB
Short interfering RNA (siRNA)
(19 base pairs)
2’,5’ oligoadenylate
synthetase
2’,5’-AS
Rnase L
Non-specific effects
Specific mRNA degradation
All mRNA are degraded
Protein synthesis is inhibited
(interference RNA)
Dykxhoorn DM, Novina CD, Sharp PA (2002)
Nature Mol Cell Biol 4: 457-467
Amplification ?
 The active molecule is
short double strand RNAs
 High specificity in siRNA
recognition
 Highly efficient (a few
molecules per cells only)
 In some organisms,
interference RNA are
amplified and carried from
cell to cell
HIV1genome
Jacque JM, Triques K, Stevenson M (2002)
Nature 418: 435-438
iRNA targets
Intracellular expression of nefGFP
Number of viral particles after infection
Brightfield
Fluorescence
T28
5’-GGUACCGAAAGCUAGGGGAUGGUU
CCACACCAACCAUCCCCUAGCUUUC
UU-3’
M28
5’-GGUACCGAAAGCUAAGGACUGGUU
CCACACCAACCAGUCCUUAGCUUUC
UU-3’
1
atggaaaaca gatggcaggt gatgattgtg tggcaagtag acaggatgag
51
gattagaaca tggaaaagtt tagtaaaaca ccatatgtat gtttcagg
ga
101
aagctagggg atggtt
t tat agacatcact atgaaagccc tcatccaaga
151
ataagttcag aagtacacat cccactaggg gatgctagat tggtaataac
201
aacatattgg ggtctgcata caggagaaag agactggcat ttgggtcagg
251
gagtctccat agaatggagg aaaaagagat atagcacaca agtagaccct
ADN sequence generating iRNA
301
gaactagcag accaactaat tcatctgtat tactttgact gtttttcaga
351
ctctgctata agaaaggcct tattaggaca catagttagc cctaggtgtg
401
aatatcaagc aggacataac aaggtaggat ctctacaata cttggcacta
451
gcagcattaa taacaccaaa aaagataaag ccacctttgc ctagtgttac
Vif = viral infection factor
501
gaaactgaca gaggatagat ggaacaagcc ccagaagacc aagggccaca
551
gagggagcca cacaatgaat ggacactag
HIV1 vif gene
T28
5’-GGUACCGAAAGCUAGGGGAUGGUU
CCACACCAACCAUCCCCUAGCUUUC
UU-3’
M28
5’-GGUACCGAAAGCUAAGGACUGGUU
CCACACCAACCAGUCCUUAGCUUUC
UU-3’
RNAi T28 folds in an hairpin structure 1
C C A
5’-GGUACCGAAAGCUAGGGGAUGGUU
C
UUCUUUCGAUCCCCUACCAAC A
C
atggaaaaca gatggcaggt gatgattgtg tggcaagtag acaggatgag
51
gattagaaca tggaaaagtt tagtaaaaca ccatatgtat gtttcagg
ga
101
aagctagggg atggtt
t tat agacatcact atgaaagccc tcatccaaga
151
ataagttcag aagtacacat cccactaggg gatgctagat tggtaataac
201
aacatattgg ggtctgcata caggagaaag agactggcat ttgggtcagg
251
gagtctccat agaatggagg aaaaagagat atagcacaca agtagaccct
ADN sequence generating iRNA
301
gaactagcag accaactaat tcatctgtat tactttgact gtttttcaga
351
ctctgctata agaaaggcct tattaggaca catagttagc cctaggtgtg
401
aatatcaagc aggacataac aaggtaggat ctctacaata cttggcacta
451
gcagcattaa taacaccaaa aaagataaag ccacctttgc ctagtgttac
Vif = viral infection factor
501
gaaactgaca gaggatagat ggaacaagcc ccagaagacc aagggccaca
551
gagggagcca cacaatgaat ggacactag
HIV1 vif gene
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