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

Structure of the lac repressor

Dimerization domain

Inducer binding domain

Operator binding domain

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

Dual control of the lac operon

glucose

lactose

CAP = catabolic gene activator protein

glucose CAPcAMP

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Structure of the CAP protein



Lac operon

Arabinose operon

tryptophanase

galactokinase

TGTGA

recognized sequences

glucose AMPc CAP

lactose

allolactose

fructose

-galactosidase

-galactosidase

-galactosidase expression

lac repressor

Non-linear regulations …





… 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’

Estradiol receptor structure

DNA binding domain

estrogen binding domain

estradiol

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

control of gene expression receptors and transcription factors inducible expression of ...

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