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

2. 12-2 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

3. 12-3 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

4. 12-4 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  -helix of the motif into the DNA major groove –Zinc fingers –Zinc modules –Modules containing 2 zinc and 6 cysteines

5. 12-5 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

6. 12-6 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

7. 12-7 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

8. 12-8 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

9. 12-9 Zinc Fingers Described by Klug in 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

10. 12-10 Arrangement of Three Zinc Fingers in a Curved Shape The zinc finger is composed of: –An antiparallel  -strand contains the 2 cysteines –2 histidines in an  -helix –Helix and strand are coordinated to a zinc ion

11. 12-11 The GAL4 Protein The GAL4 protein is a member of the zinc- containing family of DNA-binding proteins It does not have a zinc finger Each GAL4 monomer contains a DNA- binding motif with: –6 cysteines that coordinate 2 zinc ions in a bimetal thiolate cluster –Short  -helix that protrudes into the DNA major groove is the recognition module –Dimerization motif with an  -helix that forms a parallel coiled coil as it interacts with the  -helix on another GAL4 monomer

12. 12-12 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

13. 12-13 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

14. 12-14 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

15. 12-15 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

16. 12-16 Homeodomains 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

17. 12-17 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

18. 12-18 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 See that the hybrid protein still functions as an activator

19. 12-19 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

20. 12-20 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

21. 12-21 Models for Recruitment

22. 12-22 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

23. 12-23 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

24. 12-24 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

25. 12-25 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

26. 12-26 Dimerization Dimerization is a great advantage to an activator Dimerization increases the affinity between activator and its DNA target Some activators form homodimers Heterodimers are also formed –Products of the jun and fos genes form a heterodimer

27. 12-27 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

28. 12-28 Hypotheses of Enhancer Action

29. 12-29 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

30. 12-30 Control Region of the Metallothionine Gene Gene product helps eukaryotes cope with heavy metal poisoning Turned on by several different agents

31. 12-31 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

32. 12-32 An Architectural Transcription Factor Example Within 112 bp upstream of the start of transcription are 3 enhancer elements These elements bind to: –Ets-1 –LEF-1 –CREB

33. 12-33 Enhanceosome An enhanceosome is a complex of enhancer DNA with activators contacting this DNA An example is the HMG that helps to bend DNA so that it may interact with other proteins

34. 12-34 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

35. 12-35 Examples of Architectural Transcription Factors Besides LEF-1, HMG I(Y) plays a similar role in the human interferon-b control gene For the IFN-b enhancer, activation seems to require cooperative binding of several activators, including HMG I(Y) to form an enhanceosome with a specific shape

36. 12-36 Insulators Insulators act by: Enhancer-blocking activity: insulator between promoter and enhancer prevents the promoter from being activated Barrier activity: insulator between promoter and condensed, repressive chromatin prevents promoter from being repressed

37. 12-37 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

38. 12-38 Model of Multiple Insulator Action

39. 12-39 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

40. 12-40 Phosphorylation and Activation Replace this area with Figure 12.33: A model for activation of a CRE- linked gene

41. 12-41 Activation of a Nuclear Receptor-Activated Gene

42. 12-42 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

43. 12-43 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

44. 12-44 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

45. 12-45 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

46. 12-46 Three Signal Transduction Pathways

47. 12-47 Ras and Raf Signal Transduction

48. 12-48 Wnt Signaling