El papel de la curvatura de DNA en la regulación

1. El papel de la curvatura de DNA en la regulación

2. Clues and consequences of DNA bending in transcription • Nature of DNA bending. Bendability vs curved DNA • Clues of DNA bending • Inducer/ inhibitor of protein-DNA interactions • Catalyst of protein-protein interactions • DNA chaperones • Consequences of DNA bending • Channeling signals through promoter architecture • Helping response-amplification signals • Avoiding transcriptional promiscuity • DNA bending: A new signal-transduction mechanism?

3. Wedge model Structural discontinuities model Curved DNA

4. Bendability • Bendability: the ability of specific short sequences to assume preferentially conformations that accommodate the deformation associated with protein-induced bending.

5. Bendability vs curved DNA • Curved DNAis deformed even in the absence of external forces, thereby resulting in a very rigid structure. • Bendable DNA allows a mixture of many different conformational states, the equilibrium of which can be displaced toward one specific form by external forces such as proteins interacting with them.

6. Protein-mediated DNA bending • Neutralization of charges in the DNA backbone • Setting up extended protein-DNA contacts • Intercalation of protein side chains in the minor groove

7. Bend DNA is important for DNA-protein interactions • CAP:Correlation between bendability of CAP site and the affinity of the protein (Gartenberg and Crothers, 1988) • Bacterial s70-RNA polymerase induces a strong bend in the promoter upon binding (Pérez-Martín and Espinosa, 1994) • TBP and holo-TFIID bend DNA (Starr et al., 1995)

8. Clues of DNA bending in transcription • Inducer/ inhibitor of protein-DNA interactions • Catalyst of protein-protein interactions • DNA chaperones

9. DNA bending as an inducer or inhibitor of DNA-protein interactions • Structural synergy: • Pre-curved CAP DNA binding sites • HMG1 and the human progesterone receptor • Structural inhibition: • Out of phase RepA-induced bends

10. Structural synergy at the CAP-binding sites (Kahn and Crothers, 1992)

11. Structural synergy between HMG1 and PR (Oñate et al., 1994) HMG1 PR

12. RNApol RNApol Structural inhibition (Pérez-Martín and Espinosa, 1991) RNApol+ RepA RNApol

13. Clues of DNA bending in transcription • Inducer/ inhibitor of protein-DNA interactions • Catalyst of protein-protein interactions • DNA chaperones

14. Long distances: IHF histones Short distances: bendability: XylR enhancer extra factors: LEF-1 enhancer Free energy | 10 | 1000 | 100 Distance (bp) DNA bending as a catalyst of protein-protein interactions

15. XylR enhancer (Pérez-Martín and de Lorenzo, 1996) 30 bp ATP Bendable DNA

16. LEF-1 enhancer in TCRa(Giese et al., 1992) ATF/CREB LEF-1 Ets-1 PEBP2a

17. Long distances: IHF histones Short distances: bendability: XylR enhancer extra factors: LEF-1 enhancer Free energy | 10 | 1000 | 100 Distance (bp) DNA bending as a catalyst of protein-protein interactions

18. XylR s54-RNAP s54-RNAP IHF IHF at Pu promoter (Pérez-Martín and de Lorenzo, 1996) >200 bp

19. Nucleosome positioning at the Drosophila hsp26 promoter (Thomas and Elgin, 1988) -400 -300 -200 -100 HSTF-box TATA box GAGA-box GAGA-box HSTF-box RNApolII nucleosome

20. Clues of DNA bending in transcription • Inducer/ inhibitor of protein-DNA interactions • Catalyst of protein-protein interactions • DNA chaperones

21. DNA chaperones are DNA-bending proteins that stabilize an otherwise loose structure in a particular conformation which sustains the assembly of additional proteins into a higher-order complex, being displaced away from the DNA in the final assembly. DNA chaperones (Travers, 1994)

22. DNA chaperones: HMG1 and PR (Oñate et al., 1994) HMG1 PR

23. XylR s54-RNAP s54-RNAP DNA chaperones: HU at Ps promoter (Pérez-Martín and de Lorenzo, 1995) s54-RNAP HU

24. Consequences of DNA bending in transcription • Channeling signals through promoter architecture • Helping response-amplification signals • Avoiding transcriptional promiscuity

25. Co-activation CAP and MalT nucleosome in Xenopus vitellogenin B1 promoter Anti-repression CAP in ParaBAD Anti-induction YY1 in c-fos promoter IHF in nac promoter Channeling signals through promoter architecture

26. Co-activation: MalT and CAP at PmalE-PmalK in E.coli (Richet et al., 1991) PmalE RNApol CAP MalT RNApol PmalK cAMP Maltose

27. Co-activation: Xenopus vitellogenin B1 promoter (Schild et al., 1993) Cell-type Hormone NF1 HNF3 RNApolII Oestrogen receptor

28. Anti-repression: AraC and CAP at ParaBAD in E.coli (Lobell and Schleif, 1991) AraC RNApol RNApol RNApol cAMP Arabinose CAP

29. Anti-induction: YY1 at the c- fos promoter (Natesan and Gilman, 1993) cAMP YY1 RNApolII CREBP Cell status

30. Anti-induction: Nac at the nac promoter from K. aerogenes (Feng et al., 1995) Nitrogen status Nac levels NtrC Nac HU Pnac RNApol

31. Consequences of DNA bending in transcription • Channeling signals through promoter architecture • Helping response-amplification signals • Avoiding transcriptional promiscuity

32. Stimulation of lysis/lisogeny of phage Mu by DNA bending (Goosen, van de Putte, 1995) Pe O1 IHF O2 O3 O1 RNApol Pe O2 O3 DNA bending in response-amplification mechanisms LYSIS LYSOGENY

33. Consequences of DNA bending in transcription • Channeling signals through promoter architecture • Helping response-amplification signals • Avoiding transcriptional promiscuity

34. Mechanisms to suppress non-specific activation at enhancers: restrictors ? ? DNA bending and transcriptional promiscuity

35. XylR IHF s54-RNAP s54-RNAP IHF s54-RNAP -IHF +IHF Restrictor: a new role of IHF in Pu promoter (Pérez-Martín and de Lorenzo, 1995)

36. Clues and consequences of DNA bending in transcription DNA bending: A new signal-transduction mechanism?

37. Characteristics of signal-transduction mechanisms • Integration of signals • Amplification of signals • Specificity of signals

38. SignalA Kinase1 SignalB Kinase2 Kinase3 SignalC Integration of signals

39. Characteristics of signal-transduction mechanisms • Integration of signals • Amplification of signals • Specificity of signals

40. Kinase1 Kinase2 Kinase2 Kinase3 Kinase3 Kinase3 Kinase3 Amplification of signals

41. Characteristics of signal-transduction mechanisms • Integration of signals • Amplification of signals • Specificity of signals

42. SignalA SignalB Kinase1 Kinase1 Kinase2 Kinase2 Kinase3 Kinase3 Specificity of signals(Scaffolding)

43. Designing promoters “ a la carte” Specificity Amplification Integration