1. 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

2. 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

3.  -galactosidase is an inducible enzyme  -galactosidase + 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

4. The lac operon : a set of co-regulated genes  -galactosidase (z) permease (y)transacetylase (a)E.coli DNA ATGSTOPATGSTOPATGSTOP Mutants defective constitutive non-inducible z -, y -, a - i-i- Complementation Genomic DNA z +, y +, a + i-i- F’ episome z -, y +, a + i+i+  -galactosidase expression E. coli i - z + E. coli i + z - E.coli i - z + /F’i + z - -lactose+ lactose + - -+-+

5. Regulation of  -galactosidase expression by the lac repressor prepressor In the presence of the inducer In the absence of the inducer prepressor The inducer binds to the repressor, which prevents it from interacting with the operator po  -galactosidase po Binding of the repressor to the operator prevents  -galatosidase transcription

6. 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

7. Some repressor structures Trp repressor bound to its DNA operator phage  repressor bound to its DNA operator

8. 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 C O NH 2 CH 2

9. Structure of the lac repressor Dimerization domain Inducer binding domain Operator binding domain

10. The lac repressor changes its conformation upon inducer binding operatorIPTG Repressor structure in the absence of IPTG Repressor structure in the presence of IPTG

11. 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: k on = 10 10 M -1.s -1 Dissociation rate: k off = 10 -3 s -1 operator size genome size

12. Dual control of the lac operon glucose lactose

13. CAP = catabolic gene activator protein glucoseCAPcAMP Structure of the CAP protein Lac operon Arabinose operon tryptophanase galactokinase TGTGA recognized sequences

14. glucose AMPc CAP lactose allolactose fructose  -galactosidase  -galactosidase expression lac repressor Non-linear regulations …

15. … 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

16. Hydrophilic and hydrophobic inducers Hydrophilic moleculeHydrophobic molecule Second messenger production : example cAMP steroid hormones, vitamine D, retinoic acid, nitric oxide (NO) soluble hormones, neurotransmitters

17. Steroid hormones and other hydrophobic inducers Osteogenesis Male phenotype (androgen) Thyroid hormone Tadpole metamorphosis Limb bud outgrowth (anterio-posterior axis) Metabolism, inflammation Reproduction (estrogen)

18. Nuclear hormone receptor activation Glucocorticoide response element (GRE) Estrogen response element (ERE) 5’-NAGAACANNNTGTTCTN NTCTTGTNNNACAAGAN-5’ 5’-NAGGTCANNNTGACCTN NTCCAGTNNNACTGGAN-5’

19. Estradiol receptor structure DNA binding domain estrogen binding domain estradiol

20. Controlled gene knock-out by interference RNA (RNAi)  Long double stranded RNA (dsRNA) ( >30 base pairs) dsRNA-activated protein kinase PKR ATF2 elF  NF-  B 2’,5’ oligoadenylate synthetase 2’,5’ -AS Rnase L Non-specific effects All mRNA are degraded Protein synthesis is inhibited  Short interfering RNA (siRNA) (19 base pairs) Specific mRNA degradation (interference RNA)

21.  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

22. 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

23. ADN sequence generating iRNA HIV1 vif gene T28 M28 Vif = viral infection factor

24. ADN sequence generating iRNA HIV1 vif gene T28 C C C C C A A RNAi T28 folds in an hairpin structure M28 Vif = viral infection factor