general:

Activators - protein-DNA interaction

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Odd S. Gabrielsen

The sequence specific activators: transcription factors

Modular design with a minimum of two functional domains 1. DBD - DNA-binding domain 2. TAD - transactivation domain

DBD: several structural motifs classification into TF-families

TAD - a few different types Three classical categories

Acidic domains (Gal4p, steroid receptor) Glutamine-rich domains (Sp1) Proline- rich domains (CTF/NF1)

Mutational analyses - bulky hydrophobic more important than acidic

Unstructured in free state - 3D in contact with target?

Most TFs more complex Regulatory domains, ligand binding domains etc

N

C

TAD

DBD

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TF classification based on structure of DBD

bHelix-Loop-Helix(Max)

Zinc finger

Leucine zipper(Gcn4p)

p53 DBD

NFB

STATdimer

Two levels of recognition1. Shape recognition

Anhelix fits into the major groove in B-DNA. This is used in most interactions

2. Chemical recognitionNegatively charged sugar-phosphate chain involved in electrostatic interactionsHydrogen-bonding is crucial for sequence recognition

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Odd S. Gabrielsen

Alternative classification of TFs on the basis of their regulatory role Classification questions

Is the factor constitutive active or requires a signal for activation? Does the factor, once synthesized, automatically enter the nucleus to

act in transcription? If the factor requires a signal to become active in transcriptional

regulation, what is the nature of that signal?

Classification system I. Constitutive active nuclear factors II. Regulatory transcription factors

Developmental TFs Signal dependent

Steroid receptors Internal signals Cell surface receptor controlled

Nuclear Cytoplasmic

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Odd S. Gabrielsen

Classification - regulatory function

Brivanlou and Darnell (2002) Science 295, 813 -

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Sequence specific DNA-binding- essential for activators TFs create nucleation sites in promoters for

activation complexes Sequence specific DNA-binding crucial role

Principles of sequence specific DNA-binding

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Odd S. Gabrielsen

How is a sequence (cis-element) recognized from the outside?

Electrostaticinteraction

Hydrophobicinteraction

Hydrogen-bonds

Form/geometry

Shape recognition Chemical recognition

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Odd S. Gabrielsen

Complementary forms

The dimension of anhelix fits the dimensions of the major groove in B-DNA

Sidechains point outwards and are ideally positioned to engage in hydrogen bonds

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Odd S. Gabrielsen

Direct reading of DNA-sequenceRecognition of form

The dimension of an -helix fits the dimensions of the major groove in B-DNA

Most common type of interaction

Usually multiple domains participate in recognition dimers of same motif tandem repeated motif Interaction of two different motifs

recognition: detailed fit of complementary surfaces Hydration /vann participates seq specvariation of DNA-structure

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Odd S. Gabrielsen

Example

Steroid receptor

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Odd S. Gabrielsen

Recognition by complementary forms

434 fag repressor

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Odd S. Gabrielsen

DNAs form:B-DNA most common

B-form

Major groove Minor groove

wide geometryfits -helix

Each basepair with unique H-bonding-

pattern

Deep and narrow geometry

Each basepairbinary H-bonding-

pattern

B

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Odd S. Gabrielsen

DNAs form:A-form more used in RNA-binding

A-form

Major groove Minor groove

Deep and narrow geometry

Wide and shallow

A

MBV4230

Odd S. Gabrielsen

How is a sequence (cis-element) recognized from the outside?

Electrostaticinteraction

Hydrophobicinteraction

Hydrogen-bonds

Form/geometry

Shape recognition Chemical recognition

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Odd S. Gabrielsen

Next level: chemical recognition - reading of sequence information

Negatively charged sugar-phosphate chain = basis for electrostatic interaction Equal everywhere - no sequence-

recognition Still a main contributer to the

strength of binding

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Odd S. Gabrielsen

Electrostatic interactionEntropy-driven binding

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

- ------

Negative phosphate chainpartially neutralized by acloud of counter ions

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

- ------

Counter ions liberatedEntropy-driven binding

MBV4230

Odd S. Gabrielsen

How is a sequence (cis-element) recognized from the outside?

Electrostaticinteraction

Hydrophobicinteraction

Hydrogen-bonds

Form/geometry

Shape recognition Chemical recognition

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Odd S. Gabrielsen

Recognition by Hydrogen bonding

A

D A Hydrogen-bonding is a

key element in sequence specific recognition

10-20 x in contact surface

Base pairing not exhausted in duplex DNA, free positions point outwards in the major groove

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Odd S. Gabrielsen

Unexploited H-bonding possibilities in the grooves

Point outwards in major groove

Point outwards in minor groove

AT-base pair

GC-base pair

Major groove

Major groove

Minor groove

Minor groove

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Odd S. Gabrielsen

A ”bar code” in the grooves

AT-basepair

GC-basepair

Unique ”bar code” in major groove

DAA

A D A

AT-pair [AD-A] ≠ TA-pair [A-DA]GC-pair [AA-D] ≠ CG-pair [D-AA]

AT-basepair

Binary ”bar code” in minor groove

AA

GC-basepair

AAD

AT-pair [A-A] = TA-pair [A-A]GC-pair [ADA] = CG-pair [ADA]

Unique recognitionof a base pair requiresTWO hydrogen bondsIn the major groove

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Docked prot side chains exploit the H-bonding possibilities for interaction

Hydrogen-bonding is essential for sequence specific recognition 10-20 x in contact interphase Most contacts in major groove Purines most important

A Zif example

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Interaction: Protein side chain - DNA bp Close up

Amino acid sidechains points outwards from the -helix and are optimally positioned for base-interaction

Still no ”genetic code” in the form of sidechain-base rules

docking of the entire protein

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Odd S. Gabrielsen

Interaction: Protein side chain - DNA bp Close up

Amino acid sidechains points outwards from the -helix and are optimally positioned for base-interaction

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Odd S. Gabrielsen

A network of H-bonds

Example: c-Myb - DNA Protein

DNA

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Odd S. Gabrielsen

How is a sequence (cis-element) recognized from the outside?

Electrostaticinteraction

Hydrophobicinteraction

Hydrogen-bonds

Form/geometry

Shape recognition Chemical recognition

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Odd S. Gabrielsen

Hydrophobic contact points

Ile

Homeodomains

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The Homeodomain-family: common DBD-structure

Homeotic genes - biology Regulation of Drosophila development Striking phenotypes of mutants - bodyparts move Control genetic developmental program

Homeobox / homeodomain Conservered DNA-sequence “homeobox” in a

large number of genes Encode a 60 aa “homeodomain” A stably folded structure that binds DNA Similarity with prokaryotic helix-turn-helix

3D-structure determined for several HDs Drosophila Antennapedia HD (NMR) Drosophila Engrailed HD-DNA kompleks (crystal) Yeast MAT2

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Odd S. Gabrielsen

Homeodomain-family: common DBD-structure Major groove contact via a 3 -helix structure

helix 3 enters major groove (“recognition helix”) helix 1+2 antiparallel across helix 3

16 -helical aa conserved 9 in hydrophobic core some in DNA-contact interphase (common docking mechanism?)

Positions important for sequence recognition N51 invariant: H-binding Adenine, role in positioning I47 (en, Antp) hydrophobic base contact Q50 (en), S50 (2) H-bond to Adenine, determining specificity R53 (en), R54 (2): DNA-contact

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Engrailed

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Antennapedia

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Homeodomain-family: common DBD-structure

Minor groove contacted via N-terminal flexible arm R3 and R5 in engrailed and R7 in MAT2 contact AT in

minor groove R5 conserved in 97% of HDs Deletions and mutants impair DNA-binding

ftz HD (∆6aa N-term) 130-fold weaker DNA-binding MAT2 (R7A) impaired repressor POU (∆4,5) DNA-binding lost

Loop between helix 1 and 2 determines Ubx versus Antp function Close to DNA exposed for protein protein interaction

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Odd S. Gabrielsen

HD-paradox: what determines sequence specificity? Drosophila Ultrabithorax (Ubx), Antennapedia (Antp),

Deformed (Dfd) and Sex combs reduced (Scr): closely similar HD, biological rolle very different

Minor differences in DNA-binding in vitro TAAT-motif bound by most HD-factors contrast between promiscuity in vitro and specific effects in vivo

Swaps reveal that surprisingly much of the specificity is determined by the N-terminal arm which contacts the minor groove Swaps: Antp with Scr-type N-term arm shows Scr-type specificity in vivo Swaps: Dfd with Ubx-type N-term arm shows Ubx-type specificity in vivo

N-terminal arm more divergent than the rest of HD R5 and R7 (contacting DNA) are present in both Ubx, Antp, Dfd, and Scr Other tail aa diverge much more

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Odd S. Gabrielsen

Solutions of the paradox

Conformational effects mediated by N-term arm Even if the -helical HDs are very similar, a much larger diversity is found in

the N-terminal arms that contact the minor groove

Protein-protein interaction with other TFs through the N-terminal arm - enhanced affinity/specificity - the basis of combinatorial control MAT2 interaction with MCM1 - cooperative interactions Ultrabithorax- Extradenticle in Drosophila Hox-Pbx1 in mammals

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Odd S. Gabrielsen

Combinatorial TFs give enhaced specificity TFs encoded by the the

homeotic (Hox) genes govern the choice between alternative developmental pathways along the anterior–posterior axis.

Hox proteins, such as Drosophila Ultrabithorax, have low DNA-binding specificity by themselves but gain affinity and specificity when they bind together with the homeoprotein Extradenticle (or Pbx1 in mammals).

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Odd S. Gabrielsen

N-tail in protein-protein interaction- adopt different conformations

Mat-2/Mcm-1HD

HD

Conformation determinedby prot prot interaction

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Odd S. Gabrielsen

It works impressively well

Hox genes

POU family

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Odd S. Gabrielsen

POU-family: common DBD-structure

The POU-name : Pit-1 pituitary specific TF Oct-1 and Oct-2 lymphoide TFs Unc86 TF that regulates neuronal development in C.elegans

A bipartite160 aa homeodomain-related DBD a POU-type HD subdomain (C-terminally located) et POU-specific subdomain (N-terminally located) Coupled by a variabel linker (15-30 aa)

POU is a structurally bipartite motif that arose by the fusion of genes encoding two different types of DNA-binding domain.

MBV4230

Odd S. Gabrielsen

POU: Two independent subdomains

POUHD subdomain 60 aa closely similar to the classical HD Only weakly DNA-binding by itself (<HD) contacts 3´-half site (Oct-1: ATGCAAAT) docking similar to engrailed. Antp etc Main contribution to non-specific backbone

contacts

POUspec subdomain 75 aa POU-specific domain enhances DNA-affinity 1000x contacts 5´-half site (Oct-1: ATGCAAAT) contacts opposite side of DNA relative to HD structure similar to prokaryotic - and 434-

repressors

The two-part DNA-binding domain partially encircles the DNA.

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Odd S. Gabrielsen

Flexible DNA-recognition

POU-domains have intrinsic conformational flexibility and this feature appears

to confer functional diversity in DNA-recognition

The subdomains are able to assume a variety of conformations, dependent on the DNA element.

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Odd S. Gabrielsen

A POU prototype: Oct-1

Ubiquitously expressed Oct-1 (≠ cell type specific Oct-2) Oct-1 performs many divergent roles in cellular trx

regulation partly owing to its flexibility in DNA binding and ability to associate with

multiple and varied co-regulators

Oct-1 activates transcription of genes that are involved in basic cellular processes Oct-1 activates small nuclear RNA (snRNA) and S-phase histone H2B gene transcription cell-specific promoters, particularly in the immune and nervous systems immunoglobulin (Ig) heavy- and lightchains

Activate target genes by bidning to the “octamer” cis-element ATGCAAAT Hence the name “Octamer-motif binding protein”

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Odd S. Gabrielsen

Flexibility

On the natural high-affinity Oct-1 octamer (ATGCAAAT) binding site, the two Oct-1 POU-subdomains lie on opposite sides of the DNA

The unstructured linker permits flexible subdomain positioning and hence diversity in Oct-1 sequence recognition.

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Odd S. Gabrielsen

Oct-1: associates with multiple and varied co-regulators Oct-1 associates with a B-cell specific co-

regulator OCA-B (OBF-1). OCA-B stabilizes Oct-1 on DNA and provides a transcriptional activation domain. B-cell specific activation of immunoglobulin genes - for long a paradox Depended on octamer cis-elements B-cell express both ubiquitous Oct-1 and the cell type specific Oct-2

Hypothesis: Oct-2 aktivates IgGs (Wrong!) oct-2 deficient mouse normal development of early B-cells and cell

lines without Oct-2 produce abundant amounts of Ig A B-cell specific coactivator mediates Oct-1 transactivation

VP16 - a virus strategy to exploit a host TF

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Odd S. Gabrielsen

Many viruses use Oct-1 to promote infection When herpes simplex virus

(HSV) infects human cells, a virion protein called VP16, forms a trx regulatory complex with Oct-1 and the cell-proliferation factor HCF-1

VP16 = a strong transactivator, not itself DNA-binding, but becomes associated with DNA through Oct-1

The specificity of Oct-1 is altered from Octamer-seq to the virus cis-element TAATGARAT

The VP16-induced complex has served as a model for combinatorial mechanisms of trx regulation

Pax family

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Odd S. Gabrielsen

Pax family

Paired domain

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Odd S. Gabrielsen

Paired domain DBD

Major grooveinteraction:

Minor grooveinteraction:

Major grooveinteraction:

Flex?

PAI

RED

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Odd S. Gabrielsen

Pax5 - activator and repressor