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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 - PowerPoint PPT Presentation
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general:
Activators - protein-DNA interaction
MBV4230
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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Odd S. Gabrielsen
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
MBV4230
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
MBV4230
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
MBV4230
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
MBV4230
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
MBV4230
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
MBV4230
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
MBV4230
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
MBV4230
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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
MBV4230
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
MBV4230
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
MBV4230
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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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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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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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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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”
MBV4230
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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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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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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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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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Pax5 - activator and repressor