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El papel de la curvatura de DNA en la regulación

El papel de la curvatura de DNA en la regulación. 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

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El papel de la curvatura de DNA en la regulación

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  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

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