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

• Consequences of DNA bending– Channeling signals through promoter architecture– Helping response-amplification signals– Avoiding transcriptional promiscuity

• DNA bending: A new signal-transduction mechanism?

Curved DNA

Wedge model Structural discontinuitiesmodel

Bendability

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

Bendability vs curved DNA

• Curved DNA is 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.

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

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 70-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)

Clues of DNA bending in transcription

• Inducer/ inhibitor of protein-DNA interactions• Catalyst of protein-protein interactions• DNA chaperones

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

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

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

HMG1

PR

Structural inhibition (Pérez-Martín and Espinosa, 1991)

RNApolRNApol

RNApolRNApol+ RepA

Clues of DNA bending in transcription

• Inducer/ inhibitor of protein-DNA interactions• Catalyst of protein-protein interactions• DNA chaperones

DNA bending as a catalyst of protein-protein interactions

• Long distances: – IHF– histones

• Short distances:– bendability: XylR

enhancer– extra factors: LEF-

1 enhancer

Distance (bp)

|10

|100

|1000

Free

ene

rgy

XylR enhancer (Pérez-Martín and de Lorenzo, 1996)

30 bp

ATP

Bendable DNA

LEF-1 enhancer in TCR (Giese et al., 1992)

LEF-1

PEBP2

Ets-1

ATF/CREB

DNA bending as a catalyst of protein-protein interactions

• Long distances: – IHF– histones

• Short distances:– bendability: XylR

enhancer– extra factors: LEF-

1 enhancer

Distance (bp)

|10

|100

|1000

Free

ene

rgy

IHF at Pu promoter (Pérez-Martín and de Lorenzo, 1996)

XylR54-RNAP

54-RNAPIHF

>200 bp

Nucleosome positioning at the Drosophila hsp26 promoter (Thomas

and Elgin, 1988)

TATA boxHSTF-box GAGA-boxGAGA-box HSTF-box

-100-200-300-400

RNApolIInucleosome

Clues of DNA bending in transcription

• Inducer/ inhibitor of protein-DNA interactions• Catalyst of protein-protein interactions• DNA chaperones

DNA chaperones (Travers, 1994)

• 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: HMG1 and PR (Oñate et al., 1994)

PR HMG1

DNA chaperones: HU at Ps promoter (Pérez-Martín and de Lorenzo, 1995)

54-RNAPXylR

54-RNAPHU

54-RNAP

Consequences of DNA bending in transcription

• Channeling signals through promoter architecture

• Helping response-amplification signals• Avoiding transcriptional promiscuity

Channeling signals through promoter architecture

• 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

cAMP

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

PmalE

PmalK

RNApol

RNApol

MalTCAP

Maltose

Co-activation: Xenopus vitellogenin B1 promoter (Schild et al., 1993)

Oestrogenreceptor

RNApolIIHNF3NF1Hormone

Cell-type

Anti-repression: AraC and CAP at ParaBAD in E.coli (Lobell and Schleif,

1991)

RNApol

RNApol RNApol

AraC

CAPcAMP Arabinose

Anti-induction: YY1 at the c- fos promoter (Natesan and Gilman, 1993)

CREBP

YY1RNApolII

cAMP

Cell status

Anti-induction: Nac at the nac promoter from K. aerogenes (Feng et al., 1995)

PnacRNApol

HU

Nac

NtrC

Nitrogenstatus Nac levels

Consequences of DNA bending in transcription

• Channeling signals through promoter architecture

• Helping response-amplification signals• Avoiding transcriptional promiscuity

DNA bending in response-amplification

mechanisms

Stimulation of lysis/lisogeny of phage Mu by DNA bending (Goosen, van de Putte, 1995)

O1 IHF O2 O3Pe

RNApol Pe

O1

O2 O3LYSIS LYSOGENY

Consequences of DNA bending in transcription

• Channeling signals through promoter architecture

• Helping response-amplification signals• Avoiding transcriptional promiscuity

DNA bending and transcriptional promiscuity

• Mechanisms to suppress non-specific activation at enhancers: restrictors

? ?

Restrictor: a new role of IHF in Pu promoter (Pérez-Martín and de Lorenzo,

1995)XylR

IHF54-RNAP

IHF54-RNAP

+IHF54-RNAP

-IHF

Clues and consequences of DNA bending in transcription

DNA bending: A new signal-transduction mechanism?

Characteristics of signal-transduction mechanisms

• Integration of signals• Amplification of signals• Specificity of signals

Integration of signals

Kinase3

Kinase2

Kinase1SignalA

SignalB

SignalC

Characteristics of signal-transduction mechanisms

• Integration of signals• Amplification of signals• Specificity of signals

Amplification of signals

Kinase2

Kinase1

Kinase2

Kinase3Kinase3 Kinase3Kinase3

Characteristics of signal-transduction mechanisms

• Integration of signals• Amplification of signals• Specificity of signals

Specificity of signals(Scaffolding)

Kinase3

Kinase2

Kinase1

SignalA SignalB

Kinase3

Kinase2

Kinase1

Designing promoters “ a la carte”

Integration

AmplificationSpecificity