1. general: Activators - protein-DNA interaction

2. 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

3. 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

4. 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

5. Classification - regulatory function
Brivanlou and Darnell (2002) Science 295, 813 -

6. Sequence specific DNA-binding - essential for activators
TFs create nucleation sites in promoters for activation complexes
Sequence specific DNA-binding crucial role

7. Principles of sequence specific DNA-binding

8. How is a sequence (cis-element) recognized from the outside?
Shape recognition
Chemical recognition
Electrostatic
interaction
Form/
geometry
Hydrogen-
bonds
Hydrophobic
interaction

9. 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

10. Direct reading of DNA-sequence Recognition 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

11. Example
Steroid receptor

12. Recognition by complementary forms
434 fag repressor

13. DNAs form: B-DNA most common
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

14. DNAs form: A-form more used in RNA-binding
Major groove
Minor groove
Deep and narrow
geometry
Wide and shallow

15. How is a sequence (cis-element) recognized from the outside?
Shape recognition
Chemical recognition
Electrostatic
interaction
Form/
geometry
Hydrogen-
bonds
Hydrophobic
interaction

16. 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

17. Electrostatic interaction Entropy-driven binding
Na+ -
Counter ions liberated
Entropy-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

18. How is a sequence (cis-element) recognized from the outside?
Shape recognition
Chemical recognition
Electrostatic
interaction
Form/
geometry
Hydrogen-
bonds
Hydrophobic
interaction

19. 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 D A A

20. Unexploited H-bonding possibilities in the grooves
AT-base pair
GC-base pair
Major groove
Minor groove
Point outwards in major groove
Point outwards in minor groove

21. A ”bar code” in the grooves
AT-basepair
GC-basepair
Unique ”bar code”
in major groove 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 A
GC-basepair D
AT-pair [A-A] = TA-pair [A-A]
GC-pair [ADA] = CG-pair [ADA]
Unique recognition
of a base pair requires
TWO hydrogen bonds
In the major groove

22. 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

23. 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

24. Interaction: Protein side chain - DNA bp
Close up
Amino acid sidechains points outwards from the a-helix and are optimally positioned for base-interaction

25. A network of H-bonds
Example:
c-Myb - DNA
Protein
DNA

26. How is a sequence (cis-element) recognized from the outside?
Shape recognition
Chemical recognition
Electrostatic
interaction
Form/
geometry
Hydrogen-
bonds
Hydrophobic
interaction

27. Hydrophobic contact points
Ile

28. Homeodomains

29. 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

30. 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

31. Engrailed

32. Antennapedia

33. 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 Ubx versus Antp function
Close to DNA
exposed for protein protein interaction

34. 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

35. 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

36. 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).

37. N-tail in protein-protein interaction - adopt different conformationsHD b a HD
Conformation determined
by prot prot interaction
Mat-a2/Mcm-1

38. It works impressively well
Hox genes

39. POU family

40. 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.

41. 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.

42. 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.

43. 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”

44. 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.

45. 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

46. 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

47. Pax family

48. Pax family
Paired domain

49. Paired domain DBD RED Flex? PAI Major groove interaction: Minor groove

50. Pax5 - activator and repressor