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general: Activators - protein-DNA interaction. 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

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General activators protein dna interaction

general:Activators - protein-DNA interaction


The sequence specific activators transcription factors
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

DBD

N

TAD

C


Tf classification based on structure of dbd
TF classification based on structure of DBD

  • Two levels of recognition

  • 1. Shape recognition

    • Anhelix fits into the major groove in B-DNA. This is used in most interactions

  • 2. Chemical recognition

    • Negatively charged sugar-phosphate chain involved in electrostatic interactions

    • Hydrogen-bonding is crucial for sequence recognition

bHelix-Loop-Helix

(Max)

Zinc finger

Leucine zipper

(Gcn4p)

p53 DBD

NFkB

STAT

dimer


Alternative classification of tfs on the basis of their regulatory role
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


Classification regulatory function
Classification - regulatory function

Brivanlou and Darnell (2002) Science295, 813 -


Sequence specific dna binding essential for activators
Sequence specific DNA-binding- essential for activators

  • TFs create nucleation sites in promoters for activation complexes

  • Sequence specific DNA-binding crucial role



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

Shape recognition

Chemical recognition

Electrostatic

interaction

Form/

geometry

Hydrogen-

bonds

Hydrophobic

interaction


Complementary forms
Complementary forms

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

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


Direct reading of dna sequence recognition of form
Direct reading of DNA-sequenceRecognition of form

  • The dimension of an a-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


Example
Example

  • Steroid receptor


Recognition by complementary forms
Recognition by complementary forms

434 fag repressor


Dnas form b dna most common
DNAs form:B-DNA most common

B

B-form

Major groove

Minor groove

wide geometry

fits a-helix

Each basepair with

unique H-bonding-

pattern

Deep and narrow

geometry

Each basepair

binary H-bonding-

pattern


Dnas form a form more used in rna binding
DNAs form:A-form more used in RNA-binding

A

A-form

Major groove

Minor groove

Deep and narrow

geometry

Wide and shallow


How is a sequence cis element recognized from the outside1
How is a sequence (cis-element) recognized from the outside?

Shape recognition

Chemical recognition

Electrostatic

interaction

Form/

geometry

Hydrogen-

bonds

Hydrophobic

interaction


Next level chemical recognition reading of sequence information
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


Electrostatic interaction entropy driven binding

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

-

Na+

Na+

-

-

-

-

Na+

Na+

-

Na+

-

Na+

Na+

Na+

Na+

Na+

Counter ions liberated

Entropy-driven binding

Na+

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 chain

partially neutralized by a

cloud of counter ions


How is a sequence cis element recognized from the outside2
How is a sequence (cis-element) recognized from the outside?

Shape recognition

Chemical recognition

Electrostatic

interaction

Form/

geometry

Hydrogen-

bonds

Hydrophobic

interaction


Recognition by hydrogen bonding

D

A

A

Recognition by Hydrogen bonding

  • 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


Unexploited h bonding possibilities in the grooves

Major groove

Minor groove

AT-base pair

Major groove

GC-base pair

Minor groove

Unexploited H-bonding possibilities in the grooves

Point outwards in major groove

Point outwardsin minor groove


A bar code in the grooves

Unique ”bar code”

in major groove

Binary ”bar code”

in minor groove

AT-basepair

AT-basepair

GC-basepair

GC-basepair

AT-pair [AD-A] ≠ TA-pair [A-DA]

GC-pair [AA-D] ≠ CG-pair [D-AA]

D

D

AT-pair [A-A] = TA-pair [A-A]

GC-pair [ADA] = CG-pair [ADA]

A

A

A

A

D

A

A

A

A

A ”bar code” in the grooves

Unique recognition

of a base pair requires

TWO hydrogen bonds

In the major groove


Docked prot side chains exploit the h bonding possibilities for interaction
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


Interaction protein side chain dna bp
Interaction: Protein side chain - DNA bp

  • Close up

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

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

      • docking of the entire protein


Interaction protein side chain dna bp1
Interaction: Protein side chain - DNA bp

  • Close up

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


A network of h bonds
A network of H-bonds

  • Example:

  • c-Myb - DNA

Protein

DNA


How is a sequence cis element recognized from the outside3
How is a sequence (cis-element) recognized from the outside?

Shape recognition

Chemical recognition

Electrostatic

interaction

Form/

geometry

Hydrogen-

bonds

Hydrophobic

interaction




The homeodomain family common dbd structure
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 MAT2


Homeodomain family common dbd structure
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




Homeodomain family common dbd structure1
Homeodomain-family: common DBD-structure

  • Minor groove contacted via N-terminal flexible arm

    • R3 and R5 in engrailed and R7 in MAT2 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

      • MAT2 (R7A) impaired repressor

      • POU (∆4,5) DNA-binding lost

  • Loop between helix 1 and 2 determines Ubxversus Antp function

    • Close to DNA

    • exposed for protein protein interaction


Hd paradox what determines sequence specificity
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


Solutions of the paradox
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

    • MAT2 interaction with MCM1 - cooperative interactions

    • Ultrabithorax- Extradenticle in Drosophila

    • Hox-Pbx1 in mammals


Combinatorial tfs give enhanced specificity
Combinatorial TFs give enhanced 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).


N tail in protein protein interaction adopt different conformations

a

b

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

HD

HD

Conformation determined

by prot prot interaction

Mat-a2/Mcm-1


The partner may also be a linker histone
The partner may also be a linker histone

  • Repression of the mouse MyoD gene by the linker histone H1b and the homeodomain protein Msx1.

  • The first evidence that a linker histone subtype operates in a gene-specific fashion to regulate tissue differentiation




Pou family common dbd structure
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.


Pou two independent subdomains
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.


Flexible dna recognition
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.


A pou prototype oct 1
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”


Flexibility
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.


Oct 1 associates with multiple and varied co regulators
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


Many viruses use oct 1 to promote infection
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 family1
Pax family

Paired domain


Paired domain dbd
Paired domain DBD

RED

Major groove

interaction:

Minor groove

interaction:

Flex?

Major groove

interaction:

PAI


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