1. Molecular Biology Fifth Edition
Lecture PowerPoint to accompany
Molecular Biology Fifth Edition
Robert F. Weaver
Chapter 12
Transcription Activators in Eukaryotes
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

2. Transcription Activators of Eukaryotes
The general transcription factors by themselves dictate the starting point and direction of transcription but they are capable of sponsoring only a low level of transcription or basal transcription
Transcription of active genes in cells rises above the basal level
Eukaryotic cells have additional, gene-specific transcription factors called activators that bind to DNA elements called enhancers to provide the extra needed boost to transcription

3. 12.1 Categories of Activators
Activators can stimulate or inhibit transcription by RNA polymerase II
Structure is composed of at least 2 functional domains
DNA-binding domain
Transcription-activation domain
Many also have a dimerization domain

4. DNA-Binding Domains
Protein domain is an independently folded region of a protein
DNA-binding domains have DNA-binding motif
Part of the domain having characteristic shape specialized for specific DNA binding
Most DNA-binding motifs fall into 3 classes; zinc-containing modules, homeodomains and bZIP and bHLH motifs

5. Zinc-Containing Modules
There are at least 3 kinds of zinc-containing modules that act as DNA-binding motifs
All use one or more zinc ions to create a shape to fit an a-helix of the motif into the DNA major groove
Zinc fingers
Zinc modules
Modules containing 2 zinc and 6 cysteines

6. Homeodomains These domains contain about 60 amino acids
Resemble the helix-turn-helix proteins in structure and function
Found in a variety of activators
Originally identified in homeobox proteins regulating fruit fly development

7. bZIP and bHLH Motifs
A number of transcription factors have a highly basic DNA-binding motif linked to protein dimerization motifs
Leucine zippers
Helix-loop-helix
Examples include:
CCAAT/enhancer-binding protein
MyoD protein

8. Transcription-Activating Domains
Most activators have one of these domains
Some have more than one
Acidic domains such as yeast GAL4 with 11 acidic amino acids out of 49 amino acids in the domain
Glutamine-rich domains include Sp1 having 2 that are 25% glutamine
Proline-rich domains such as CTF which has a domain of 84 amino acids, 19 proline

9. Summary
Eukaryotic activators are composed of at least two domains: a DNA-binding domain and a transcription-activating domain
DNA-binding domains contain motifs such as zinc modules, homeodomains, and bZIP or bHLH motifs
Transcription activating domains can be acidic, glutamine-rich or proline-rich

10. 12.2 Structures of the DNA-Binding Motifs of Activators
DNA-binding domains have well-defined structures
X-ray crystallographic studies have shown how these structures interact with their DNA targets
Interaction domains forming dimers, or tetramers, have also been described
Most classes of DNA-binding proteins can’t bind DNA in monomer form

11. Zinc Fingers Described by Klug in GTF TFIIIA
Nine repeats of a 30-residue element:
2 closely spaced cysteines followed 12 amino acids later by 2 closely spaced histidines
Coordination of amino acids to the metal helps form the finger-shaped structure
Rich in zinc, enough for 1 zinc ion per repeat
Specific recognition between the zinc finger and its DNA target occurs in the major groove

12. Arrangement of Three Zinc Fingers in a Curved Shape
The zinc finger is composed of:
An antiparallel b-strand that contains 2 cysteines
2 histidines in an a-helix
Helix and strand are coordinated to a zinc ion

13. The GAL4 Protein
The GAL4 protein is a member of the zinc-containing family of DNA-binding proteins
Each GAL4 monomer contains a DNA-binding motif with:
6 cysteines that coordinate 2 zinc ions in a bimetal thiolate cluster
Short a-helix that protrudes into the DNA major groove is the recognition module
Dimerization motif with an a-helix that forms a parallel coiled coil as it interacts with the a-helix on another GAL4 monomer

14. The Nuclear Receptors
A third class of zinc module is the nuclear receptor
This type of protein interacts with a variety of endocrine-signaling molecules
Protein plus endocrine molecule forms a complex that functions as an activator by binding to hormone response elements and stimulating transcription of associated genes

15. Type I Nuclear Receptors
These receptors reside in the cytoplasm bound to another protein
When receptors bind to their hormone ligands:
Release their cytoplasmic protein partners
Move to nucleus
Bind to enhancers
Act as activators

16. Glucocorticoid Receptors
DNA-binding domain with 2 zinc-containing modules
One module has most DNA-binding residues
Other module has the surface for protein-protein interaction to form dimers

17. Types II and III Nuclear Receptors
Type II nuclear receptors stay within the nucleus bound to target DNA sites
Without ligands the receptors repress gene activity
When receptors bind ligands, they activate transcription
Type III receptors are “orphan” whose ligands are not yet identified

18. Homeodomain-DNA Complex
Homeodomains contain DNA-binding motif functioning as helix-turn-helix motifs
A recognition helix fits into the DNA major groove and makes specific contacts there
N-terminal arm nestles in the adjacent minor groove

19. The bZIP and bHLH Domains
bZIP proteins dimerize through a leucine zipper
This puts the adjacent basic regions of each monomer in position to embrace DNA target like a pair of tongs
bHLH proteins dimerize through a helix-loop-helix motif
Allows basic parts of each long helix to grasp the DNA target site
bHLH and bHLH-ZIP domains bind to DNA in the same way, later have extra dimerization potential due to their leucine zippers

20. 12.3 Independence of the Domains of Activators
DNA-binding and transcription-activating domains of activator proteins are independent modules
Making hybrid proteins with DNA-binding domain of one protein, transcription-activating domain of another
The hybrid protein still functions as an activator

21. 12.4 Functions of Activators
Bacterial core RNA polymerase is incapable of initiating meaningful transcription
RNA polymerase holoenzyme can catalyze basal level transcription
Often insufficient at weak promoters
Cells have activators to boost basal transcription to higher level in a process called recruitment

22. Eukaryotic Activators
Eukaryotic activators also recruit RNA polymerase to promoters
Stimulate binding of general transcription factors and RNA polymerase to a promoter
2 hypotheses for recruitment:
General TF cause a stepwise build-up of preinitiation complex
General TF and other proteins are already bound to polymerase in a complex called RNA polymerase holoenzyme

23. Models for Recruitment of Preinitiation Complex Components in Yeast

24. Recruitment of TFIID
Acidic transcription-activating domain of the herpes virus transcription factor VP16 binds to TFIID under affinity chromatography conditions
TFIID is rate-limiting for transcription in some systems
TFIID is the important target of the VP16 transcription-activating domain

25. Recruitment of the Holoenzyme
Activation in some yeast promoters appears to function by recruitment of holoenzyme
This is an alternative to the recruitment of individual components of the holoenzyme one at a time
Some evidence suggests that recruitment of the holoenzyme as a unit is not common

26. Recruitment Model of GAL11P-containing Holoenzyme
Dimerization domain of FAL4 binds to GAL11P in the holoenzyme
After dimerization, the holoenzyme, along with TFIID, binds to the promoter, activating the gene

27. 12.5 Interaction Among Activators
General transcription factors must interact to form the preinitiation complex
Activators and general transcription factors also interact
Activators usually interact with one another in activating a gene
Individual factors interact to form a protein dimer facilitating binding to a single DNA target site
Specific factors bound to different DNA target sites can collaborate in activating a gene

28. Dimerization
Dimerization is a great advantage to an activator as it increases the affinity between the activator and its DNA target
Some activators form homodimers but others function as heterodimers

29. Action at a Distance
Bacterial and eukaryotic enhancers stimulate transcription even though located some distance from their promoters
Four hypotheses attempt to explain the ability of enhancers to act at a distance
Change in topology
Sliding
Looping
Facilitated tracking

30. Hypotheses of Enhancer Action

31. 3C: Method to detect DNA looping
Chromosome conformation capture (3C) is a technique used to determine if enhancer action requires DNA looping
Used to test whether two remote DNA regions, such as an enhancer and a promoter, are brought together

32. Genomic Imprinting
Because most eukaryotes are diploid organisms, you would predict that it does not matter which allele of any given gene came form the mother or the father
This is true in most cases but there are important exceptions
The differences between the genes resides in how they are modified, or imprinted, differently in females and males
Evidence exists in mice and humans

33. Transcription Factories
Discrete nuclear sites where transcription of multiple genes occurs
If two or more genes on the same chromosome are clustered in the same transcription factory, DNA loops would naturally form between them
Thus, the existence of transcription factories implies the existence of DNA loops in eukaryotic cells

34. Evidence for Transcription Factories
Cook and colleagues counted the number of transcription factories by labeling growing RNA chains in HeLa cells with BrU followed by permeabilization and further labeling with biotin-CTP detected with anti-BrU or anti-biotin primary antibodies followed by secondary antibodies labeled with gold particles
They concluded that transcription is associated with the clusters, not the single particles

35. Complex Enhancers
Many genes can have more than one activator-binding site permitting them to respond to multiple stimuli
Each of the activators that bind at these sites must be able to interact with the preinitiation complex assembling at the promoter, likely by looping out any intervening DNA

36. Control Region of the Metallothionine Gene
The metallothionine gene product helps eukaryotes cope with heavy metal poisoning
Turned on by several different agents
Complex enhancers enable a gene to respond differently to different combinations of activators
This gives cells exquisitely fine control over their genes in different tissues, or at different times in a developing organism

37. Architectural Transcription Factors
Architectural transcription factors are those transcription factors whose sole or main purpose seems to be to change the shape of a DNA control region so that other proteins can interact successfully to stimulate transcription

38. Example of Architectural Transcription Factor: Control region of the human TCR  chain gene
Within 112 bp upstream of the start of transcription are 3 enhancer elements
These elements bind to:
Ets-1, LEF-1, CREB

39. Enhanceosome
An enhanceosome is a nucleoprotein complex containing a collection of activators bound to an enhancer in such a way that stimulates transcription
The archetypal enhanceosome involves the IFN enhancer with a structure that involves eight polypeptides bound cooperatively to an essentially straight 55-bp strech of DNA

40. DNA Bending Aids Protein Binding
The activator LEF-1 binds to the minor groove of its DNA target through its HMG domain and induces strong bending of DNA
LEF-1 does not enhance transcription by itself
Bending it induces helps other activators bind and interact with activators and general transcription factors

41. Insulators
Insulators can shield genes from activation by enhancers (enhancer blocking activity)
Insulators can shield genes from repression by silencers (barrier activity)

42. Mechanism of Insulator Activity
Sliding model
Activator bound to an enhancer and stimulator slides along DNA from enhancer to promoter
Looping model
Two insulators flank an enhancer, when bound they interact with each other isolating enhancer

43. Model of Multiple Insulator Action

44. Summary
Some insulators have both enhancer-blocking and barrier activities, but some have only one or the other
Insulators may do their job by working in pairs that bind proteins that can interact to form DNA loops that would isolate enhancers and silencers so they can no longer stimulate or repress promoters
Insulators may establish boundaries between DNA regions in a chromosome

45. 12.6 Regulation of Transcription Factors
Phosphorylation of activators can allow them to interact with coactivators that in turn stimulate transcription
Ubiquitylation of transcription factors can mark them for
Destruction by proteolysis
Stimulation of activity
Sumoylation is the attachment of the polypeptide SUMO which can target for incorporation into compartments of the nucleus
Methylation and acetylation can modulate activity

46. Phosphorylation and Activation: A Model for activation of a CRE-linked gene
Replace this area with Figure 12.33:
A model for activation of a CRE-linked gene

47. Model for the Activation of a Nuclear Receptor-Activated Gene

48. Ubiquitylation
Ubiquitylation, especially monoubiquitylation, of some activators can have an activating effect
Polyubiquitylation marks these same proteins for destruction
Proteins from the 19S regulatory particle of the proteasome can stimulate transcription

49. Activator Sumoylation
Sumoylation is the addition of one or more copies of the 101-amino acid polypeptide SUMO (Small Ubiquitin-Related Modifier) to lysine residues on a protein
Process is similar to ubiquitylation
Results quite different – sumoylated activators are targeted to a specific nuclear compartment that keeps them stable

50. Activator Acetylation
Nonhistone activators and repressors can be acetylated by HATs
HAT is the enzyme histone acetyltransferase which can act on nonhistone activators and repressors
Such acetylation can have either positive or negative effects

51. Signal Transduction Pathways
Signal transduction pathways begin with a signaling molecule interacting with a receptor on the cell surface
This interaction sends the signal into the cell and frequently leads to altered gene expression
Many signal transduction pathways rely on protein phosphorylation to pass the signal from one protein to another
This leads to signal amplification at each step

52. Three pathways that use CBP/p300 to mediate transcription activation

53. Ras and Raf Signal Transduction