1. Chapt 12 Transcription Activators in Eukaryotes
Student learning outcomes:
Explain how gene-specific activators binding to DNA elements (enhancers) activate transcription.
Activators contain at least 2 domains: DNA-binding domain (DBD) and transcription-activation domain (TAD).
Transcription factor p53
(tumor suppressor) binding DNA

2. Describe some DBD and TAD motifs:
Zinc modules,
homeodomain, bZIP, bHLH (shared with prokaryotes)
Examples of activators
Explain how activators function by contacting GTFs, recruiting pol II or contacting coactivators
Describe how activators and coactivators can be controlled by modifications including:
ubiquityation, sumoylation, phosphorylation, acetylation
Important Figures: 1, 2*, 3, 4*, 6, 9, 10*, 12*,16, 18*, 20, 23, 24, 27, 33*, 34*, 35, 36, 37; Table 1
Review questions: 1, 2, 3, 4, 5, 6, 9, 10, 13, 14, 17*, 18, 24, 28, 30, 31; Analy Q 1

3. 12.1 Categories of Activators
Activators can stimulate or inhibit transcription by RNA polymerase II
Composed of at least 2 functional domains:
DNA-binding domain (DBD)
Transcription-activation domain (TAD)
Many also have dimerization domain
Domain - independently folded region of protein
Ex. Fig Gal4 activator of yeast

4. DNA-Binding Domains (DBD)
DNA-binding domains - DNA-binding motifs:
3 Characteristic shapes for specific DNA binding
Zinc-containing modules: use Zinc ions to create shape to fit a-helix of motif into DNA major groove
Zn fingers, Zn modules, Modules with 2 Zn and 6 cys
Homeodomains: about 60 amino acids - resemble HTH (helix-turn-helix) proteins in structure and function;
ex. homeobox proteins regulate fruit fly development
bZIP and bHLH motifs: highly basic DNA-binding motif linked to protein dimerization motifs
Leucine zippers, Helix-loop-helix (ex. CCAAT enhancer-binding protein)

5. Transcription-Activating Domains (TAD)
Most activators have at least one of these domains:
Acidic domains – ex. yeast GAL4 (11 acidic amino acids out of 49 amino acids) in domain
Glutamine-rich domains – ex. Sp1 has 2 regions that are 25% glutamine (Gln)
Proline-rich domains – ex. CTF has domain with 19 proline in 84 amino acids

6. 12.2 Structures of DNA-Binding Motifs of Activators
DNA-binding domains well-defined structures (Ch. 9)
X-ray crystallography shows how structures interact with DNA targets
Interaction domains often form dimers, or tetramers,
Most DNA-binding proteins can’t bind DNA in monomer form
Compare common DNA binding motif in prokaryotes

7. Helix-Turn-Helix motif is common: lac, trp, l, other phage repressors
l Family of Repressors Repressors have recognition helices (red) fit major groove of operator
Helix-turn-helix motif (HTH)
Specificity of binding depends on amino acids in recognition helices
Fig. 9.2

8. High-Resolution Analysis of l Repressor-Operator Interactions
Structural Features from co-crystal:
Recognition helices of repressor monomer (3, red) nestle into DNA major grooves in 2 half-sites
Helices approach, hold two monomers together in repressor dimer
DNA similar in shape to B-form DNA
DNA bends at two ends of fragment as it curves around repressor dimer
Figs. 9.6, 9.7:
Repressor dimer binding to symmetric operator O1

9. Eukaryotic Homeodomains have HTH motif
Homeodomains contain DNA-binding HTH (helix-turn-helix) motif
Recognition helix (3) fits in DNA major groove, makes specific contacts
N-terminal arm nestles in adjacent minor groove
First identified Drosophila developmental genes –
Fig. 9 Drosophila homeotic engrailed protein binding target
Fig. 8 Antennapedia mutant has legs in place of antennae

10. Eukaryotic bZIP and bHLH Domains
bZIP proteins dimerize through leucine zipper motif
Leu every 7 aa (one face); hydrophobic interactions
adjacent basic regions embrace DNA like tongs
bHLH proteins dimerize through helix-loop-helix motif
basic parts of each long helix grasp DNA target site
Ex. bZIP yeast GCN4 activator
Fig. 10 structure leucine zipper
Fig. 11 bZIP of yeast GCN4 activator

11. Eukaryotic bZIP and bHLH Domains
bHLH motif in activator myoD:
HLH is dimerization
Long helix 1 has basic region
that binds DNA
Both bHLH and bZIP domains are found in Myc, Max oncogenes; heterodimers bind to DNA like HLH; extra dimerization potential from leu zippers

12. Zinc Fingers 9 repeats of 30-aa element: First described forTFIIIA
Fig. 1 3D shape of 1 Xenopus Zn finger:
Zn blue; Cys yellow S;
His blue
Zinc Fingers
First described forTFIIIA
9 repeats of 30-aa element:
2 closely spaced Cys followed 12 aa by 2 closely spaced His
Coordination of aa to metal helps form finger-shape
Protein rich in Zn: 1 Zn per repeat
Specific recognition between Zn finger and DNA in major groove
Fig. 2; Zn finger has antiparallel b-strand (2 Cys) and a-helix (2 His)

13. 3 Zinc Fingers in Curved Shape bind DNA
Zif268 is mouse TFIIIA-class protein
Each Zn finger:
antiparallel b-strand contains 2 Cysteines
2 Histidines in a-helix
Helix and strand coordinated to Zn ion
aa of helix recognize bp of major groove
Fig. 3

14. Zn module: GAL4 Protein of Yeast
GAL4 regulates expression of genes for galactose metabolism
Binds target UAS (upstream activating sequence)
Member of Zn-containing family of DNA-binding proteins, Not zinc finger
Each GAL4 monomer contains DNA-binding motif:
6 Cys coordinate 2 Zn ions in a bimetal thiolate cluster
DNA recognition - short a-helix protrudes into major groove
Dimerization motif on other end - a-helix forms parallel coiled coil, interacts with a-helix on other GAL4 monomer

15. Zn module - GAL4 protein of yeast
Fig. 4 dimer of GAL4 binding DNA: Zn yellow
DNA recognition aa 8 to 40; linker 41-49; dimerization 50-64

16. Zn modules - Nuclear Receptors
Nuclear hormone receptors interact with different endocrine-signaling molecules
Protein plus endocrine molecule forms complex that functions as activator:
Binds hormone response elements, stimulates transcription of associated genes
Ligands for these receptors are lipid-soluble, cross membrane (steroids, retinoic acid, vitamin D)

17. Type I Nuclear Receptors
Type 1 include Glucocorticoid (GR), Estrogen (ER)
Reside in cytoplasm bound to protein Hsp90
When receptors bind to hormone ligands:
Release cytoplasmic protein partners
Move to nucleus; dimerize
Bind to enhancers
Act as activators
Fig. 6

18. Glucocorticoid Receptor binding DNA
Fig. 6 structure with half sites separated by extra bp; only 1 module binds best
DNA-binding domain with 2 Zn-containing modules
One module has most of DNA-binding residues
Other module has surface for protein-protein interaction to form dimers

19. Types II and III Nuclear Receptors
Type II nuclear receptors stay within nucleus
Examples: thyroid hormone receptor (TR),
vitamin D receptor (D3)
Retinoic acid receptor (RAR) (more Ch. 13)
Bound to target DNA sites
Without ligands, receptors repress gene activity
When receptors bind ligands, activate transcription
Type III receptors are “orphan” whose ligands are not yet identified
All use a-helix for interaction with DNA sequences

20. 12.3 Independence of Domains of Activators
DNA-binding, transcription-activating domains of activator proteins are independent modules
Hybrid proteins with DNA-binding domain of one protein, transcription-activating domain of another
Hybrid protein still functions as activator
Fig. 18; yeast expressGal4 TAD on Gal4 or lexA DBD

21. 12.4 Functions of Activators for pol II
Eukaryotic activators recruit pol II to promoters
Stimulate binding of general transcription factors (GTFs) and pol II to promoter
2 hypotheses for recruitment:
GTF cause stepwise build-up of preinitiation complex
GTF and other proteins already bound to polymerase in complex called RNAP holoenzyme
Kornberg lab quantitation of proteins tested models

22. Models for Recruitment
Fig. 14

23. Recruitment Model of GAL11P-containing Holoenzyme
Fig. 16
Ptashne found dimerization domain of GAL4 binds to mutant form of Gal 11 (GAL11P) in holoenzyme (GAL11P is potentiator) [GAL11 is part of mediator complex]
Holoenzyme, plus TFIID, then binds to promoter, activating gene

24. Recruitment Model of GAL11P-containing Holoenzyme
Activation in some yeast promoters appears to function by recruitment of holoenzyme
chimeric activator proteins with lexA DBD and other domains recruit holoenzyme
Fig. 17

25. Recruitment of holoenzyme may not be that common
Kornberg evidence:
TAP- epitope tags on components:
Antibodies on immunoblot quantified pol II, GTFs, mediator:
pol II and some GTFs present in much higher concentration
Unlikely holoenzyme recruited as unit
Fig. 18 Pol II about 3x more than Mediator, TFIIH

26. 12.5 Interaction Among Activators
General transcription factors (GTFs) interact to form preinitiation complex (PIC)
Activators and GTFs also interact
Activators interact with one another to activate gene
Individual factors interact to form protein dimer facilitating binding to single DNA target
Specific factors bound to different DNA targets can collaborate to activate gene

27. Importance of Dimerization to Activation
Dimerization increases affinity between activator and DNA target:
Some activators form homodimers (ex. GAL4)
Some form heterodimers
Ex. Products of jun and fos
Oncogenes form heterodimer,
bind AP-1 sites; heterodimer
Binds better than homodimers
Fig. 19; EMSA assay; Fos or Fos Core mixed with Jun or Jun core and AP-1 site; M = myc oncoprotein

28. Activators can Act at a Distance
Bacterial and eukaryotic enhancers stimulate transcription even though located some distance from promoters
Hypotheses explain ability of enhancers to act at a distance
Change in topology
Sliding
Looping **
Facilitated tracking
Looping brings activators bound to different enhancers closer to promoter

29. Hypotheses of Enhancer Action
Fig. 20:
a. Topology
b. Sliding
c. Looping
d. Tracking

30. Complex Enhancers
Many genes have more than one activator-binding site, -> respond to multiple stimuli
Each activator that binds must interact with PIC assembling at promoter, likely by looping out intervening DNA
Consider whole region upstream of promoter as an enhancer (rather than each specific site for each factor); different factors binding permit fine level of control on expression
Combinatorial code: final result of all activators, inhibitors bound

31. Complex enhancer: Control Region of Metallothionein Gene
Fig. 23
Gene product helps eukaryotes cope with heavy metal poisoning (small proteins rich in cys; bind metals)
Turned on by several different agents
BLE = basal level enhancer; MRE = metal response element;
GRE = glucocorticoid response element; GC = Sp1 binding

32. Complex enhancers in development
Figs. 24, 25: sea urchin Endo16 gene: different activators and modules at different times control expression
Red = enhancers;
Blue = architectural factors
Ovals - activators

33. Architectural Transcription Factors
Architectural transcription factors: main purpose to change shape of DNA control region so other proteins bind successfully to stimulate transcription
HMG proteins bend DNA, help activators bind
(HMG, small nuclear proteins with high electrophoretic mobility)
ex. LEF-1 (T-cell receptor); HMGI (Y) for interferon B gene

34. Architectural Transcription Factor
Fig. 26 T cell receptor a chain
T cell receptor a-chain: enhancer elements upstream
Elements bind to: Ets-1 LEF-1 CREB
CREB (cyclic AMP response element binding protein)
LEF-1 (lymphoid enhancer) not enhance transcription alone
Activator LEF-1 binds minor groove of DNA through HMG domain, induces strong bending of DNA
DNA bending helps other activators bind, and interact with activators and general transcription factors

35. Enhanceosome Complex of enhancer DNA with activators contacting DNA
Ex. HMG helps bend DNA so that it interacts with other proteins
Fig. 27
HMG I(Y) plays similar role in human interferon-b control gene:
Activation seems to require cooperative binding of several activators, including HMG I(Y) to form enhanceosome with a specific shape

36. Insulators prevent activation of unrelated genes by nearby enhancers:
Fig. 28
Enhancer-blocking activity: insulator between promoter and enhancer prevents promoter activated
Barrier activity: insulator between promoter and condensed, repressive chromatin prevents promoter repressed

37. Mechanism of Insulator Activity
Sliding model
Activator bound to enhancer and stimulator slides along DNA to promoter
Looping model
Two insulators flank an enhancer; when bound, interact to isolate enhancer
Fig. 29

38. 12.6 Regulation of Transcription Factors
Hormones binding nuclear receptors caused them to move to nucleus, or to change from repressor to activator (Ch. 13)
Phosphorylation of activators allows to interact with coactivators to stimulate transcription
Ubiquitylation of transcription factors marks them for
Destruction by proteolysis
Stimulation of activity
Sumoylation - attachment of polypeptide SUMO; targets movement to compartments of nucleus
Methylation and acetylation can modulate activity

39. Coactivators mediate signals from activator to basal transcription apparatus
CBP/p300 is common coactivator:
CREB binding protein/ 300kD protein
HAT activity, acetylates histones and others
CREB (cyclic AMP responsive element binding protein) binds CRE (Fig. 33)
Nuclear receptors plus hormone bind DNA (Fig. 34)
Both effects mediated through CBP

40. Coactivators do not stimulate basal transcription, only activated
Mediator: (Kornberg)
Mediates activation; binds CTD of pol II
Found in yeast first
Many similar complexes:
SMCC/ TRAP
CBP/p300 (CREB-binding)
CRSP (for SP1)
SRC (steroid receptor co-)
Fig. 32

41. Coactivators Mediate Activation
Ex. CREB stimulates basal transcription machinery via CBP/p300
Protein Kinase A functions in signal transduction pathway
Phosphorylation of CREB by PKA is critical for activation;
Fig. 33

42. Activation of Nuclear Receptor-Activated Gene uses CBP/ SRC coactivator
CARM1 methylates proteins
Fig. 34

43. Ubiquitylation affects activation
Ubiquitin - 76-aa polypeptide covalently attached to Lys residues by Ubiquitin ligase
Ubiquitylation, especially monoubiquitylation, of activators can have activating effect
Polyubiquitylation marks proteins for destruction in proteasome, reduces or abolishes activation
Proteins from 19S regulatory particle of proteasome can stimulate transcription

44. Sumoylation affects Activation
Sumoylation - addition of one or more copies of 101aa polypeptide SUMO (Small Ubiquitin-Related Modifier) to Lysine residues; similar to ubiquitylation
Sumoylated activators targeted to a nuclear compartment that keeps them stable,
but not activating targets
(PML nuclear bodies)
Ex. PML protein in
promyelocytic leukemia;
B-catenin and LEF-1 SUMO
Some proteins in nuclear bodies

45. Acetylation affects Activators
HAT - enzyme histone acetyltransferase, modifies histone tails with acetyl groups (Ch. 13)
Activity of CBP/p300
Nonhistone activators and repressors can be acetylated by HATs
Acetylation has either positive or negative effects

46. Signal Transduction Pathways
Begins with signaling molecule interacting with receptor on cell surface (hormones, growth factors)
Interaction sends signal into cell (second messenger pathway)
Often leads to altered gene expression
Often protein phosphorylation passes signal from one protein to another (PKA, MAPK, G proteins)
Signal amplification occurs at each step

47. Three Signal Transduction Pathways that share coactivator CBP/p300

48. Review questions
1. List three different classes of DNA-binding domains found in eukaryotic transcription factors.
2. List three different classes of transcription-activation domains in eukaryotic transcription factors.
7. Explain the difference between type I and II nuclear receptors, and give an example of each.
28. Draw diagrams to illustrate action of CBP as activator of (a) phosphorylated CREB; (b) nuclear receptor