Control of gene expression Receptors and transcription factors Inducible expression of ...
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Transcript of Control of gene expression Receptors and transcription factors Inducible expression of ...
Control of gene expression
Receptors and transcription factors
Inducible expression of -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
DNA
mRNA
proteins
receptors
transciption factors
ENVIRONMENT
CELL
Receptors are proteins which trigger
intracellular chemical signaling in response to
an external stimulus
Transcription factors are proteins which
control mRNA synthesis
-galactosidase is an inducible enzyme
-ga
lact
osid
ase
+ inducer
- inducer
temps
An inducer is a molecule which modulates the level of expression of a protein
-galactosidase allows Escherichia coli to grow using lactose as its only carbon source
The lac operon : a set of co-regulated genes
-galactosidase (z) permease (y) transacetylase (a)E.coli DNA
ATG STOP ATG STOP ATG STOP
Mutants
defective
constitutive
non-inducible
z-, y-, a-
i-
Complementation
Genomic DNA z+, y+, a+i-
F’ episome z-, y+, a+i+
-galactosidase expression
E. coli i-z+
E. coli i+z-
E.coli i-z+/F’i+z-
-lactose + lactose+ +- -- +
Regulation of -galactosidase expression by the lac repressor
p repressor
In the presence of the inducer
In the absence of the inducer
p repressor
The inducer binds to the repressor, which prevents it from interacting with the operator
p o -galactosidase
p o -galactosidase
Binding of the repressor to the operator prevents -galatosidase transcription
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
Trp repressor bound to its DNA operator
phage 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
Non-specific interaction : Some amino acids of the transcription factors bind to the DNA polyphosphate chains
Asparagine
CH 2
CO
NH 2
CH 2
The lac repressor changes its conformation upon inducer binding
operator
IPTG
Repressor structure in the absence of IPTG
Repressor structure in the presence of IPTG
Dimerization of the lac repressor increases the affinity for the operator
In the absence of inductor
In the presence of inductor
Efficient capture mechanism, beyond simple diffusion
Specificity in the operator-repressor interaction
Affinity: K = 10-13 M
Association rate:
kon = 1010 M-1.s-1
Dissociation rate:
koff = 10-3 s-1
tailledel'opérateurtailledugénome
=30
5.106 =6.10−5operator size
genome size
CAP = catabolic gene activator protein
glucose CAPcAMP
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Structure of the CAP protein
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Lac operon
Arabinose operon
tryptophanase
galactokinase
TGTGA
recognized sequences
glucose AMPc CAP
lactose
allolactose
fructose
-galactosidase
-galactosidase
-galactosidase expression
lac repressor
Non-linear regulations …
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… may give rise to complex processes
bi-stability
hysteresis
oscillations
Atkinson et al. (2003) Cell 113: 597-607
Ozbudak et al. (2004) Nature 427: 737-740
Hydrophilic and hydrophobic inducers
Hydrophilic molecule Hydrophobic molecule
Second messenger production : example cAMP
steroid hormones, vitamine D, retinoic acid, nitric oxide (NO)
soluble hormones, neurotransmitters
Steroid hormones and other hydrophobic inducers
Osteogenesis
Male phenotype (androgen)
Thyroid hormoneTadpole metamorphosis
Limb bud outgrowth (anterio-posterior axis)
Metabolism, inflammation Reproduction
(estrogen)
Nuclear hormone receptor activation
Glucocorticoide response element (GRE)
Estrogen response element (ERE)
5’-NAGAACANNNTGTTCTNNTCTTGTNNNACAAGAN-5’
5’-NAGGTCANNNTGACCTNNTCCAGTNNNACTGGAN-5’
Controlled gene knock-out by interference RNA (RNAi)
Long double stranded RNA (dsRNA)( >30 base pairs)
dsRNA-activatedprotein kinase
PKR
ATF2 elF NF-B
2’,5’ oligoadenylatesynthetase2’,5’-AS
Rnase L
Non-specific effectsAll mRNA are degraded
Protein synthesis is inhibited
Short interfering RNA (siRNA)(19 base pairs)
Specific mRNA degradation(interference RNA)
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
Amplification ?
Dykxhoorn DM, Novina CD, Sharp PA (2002)Nature Mol Cell Biol 4: 457-467
HIV1genome
iRNA targets
Number of viral particles after infection
Intracellular expression of nef-GFP
Brightfield
Fluorescence
Jacque JM, Triques K, Stevenson M (2002)Nature 418: 435-438
ADN sequence generating iRNA
1atggaaaaca gatggcaggt gatgattgtg tggcaagtag acaggatgag
51gattagaaca tggaaaagtt tagtaaaaca ccatatgtat gtttcaggga
101aagctagggg atggttttat agacatcact atgaaagccc tcatccaaga
151ataagttcag aagtacacat cccactaggg gatgctagat tggtaataac
201aacatattgg ggtctgcata caggagaaag agactggcat ttgggtcagg
251gagtctccat agaatggagg aaaaagagat atagcacaca agtagaccct
301gaactagcag accaactaat tcatctgtat tactttgact gtttttcaga
351ctctgctata agaaaggcct tattaggaca catagttagc cctaggtgtg
401aatatcaagc aggacataac aaggtaggat ctctacaata cttggcacta
451gcagcattaa taacaccaaa aaagataaag ccacctttgc ctagtgttac
501gaaactgaca gaggatagat ggaacaagcc ccagaagacc aagggccaca
551gagggagcca cacaatgaat ggacactag HIV1 vif gene
5’-GGUACCGAAAGCUAGGGGAUGGUUCCACACCAACCAUCCCCUAGCUUUCUU-3’T28
5’-GGUACCGAAAGCUAAGGACUGGUUCCACACCAACCAGUCCUUAGCUUUCUU-3’M28
Vif = viral infection factor
ADN sequence generating iRNA
1atggaaaaca gatggcaggt gatgattgtg tggcaagtag acaggatgag
51gattagaaca tggaaaagtt tagtaaaaca ccatatgtat gtttcaggga
101aagctagggg atggttttat agacatcact atgaaagccc tcatccaaga
151ataagttcag aagtacacat cccactaggg gatgctagat tggtaataac
201aacatattgg ggtctgcata caggagaaag agactggcat ttgggtcagg
251gagtctccat agaatggagg aaaaagagat atagcacaca agtagaccct
301gaactagcag accaactaat tcatctgtat tactttgact gtttttcaga
351ctctgctata agaaaggcct tattaggaca catagttagc cctaggtgtg
401aatatcaagc aggacataac aaggtaggat ctctacaata cttggcacta
451gcagcattaa taacaccaaa aaagataaag ccacctttgc ctagtgttac
501gaaactgaca gaggatagat ggaacaagcc ccagaagacc aagggccaca
551gagggagcca cacaatgaat ggacactag HIV1 vif gene
5’-GGUACCGAAAGCUAGGGGAUGGUUCCACACCAACCAUCCCCUAGCUUUCUU-3’T28
5’-GGUACCGAAAGCUAGGGGAUGGUU
UUCUUUCGAUCCCCUACCAA
C C
CC C
A
A
RNAi T28 folds in an hairpin structure
5’-GGUACCGAAAGCUAAGGACUGGUUCCACACCAACCAGUCCUUAGCUUUCUU-3’M28
Vif = viral infection factor