1. Structure of the DNA-binding motifs of activators Chapter 12

2. 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. DNA-binding domains 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. 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 α-helix of the motif into the DNA major groove –Zinc fingers – TFIIIA and Sp1 –Zinc modules – Glucocorticoid receptor –Modules containing 2 zinc and 6 cysteines – GAL4

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. 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. Transcription-Activation Domains Acidic domains: GAL4 Glutamine-rich domains: Sp1 Proline-rich domains: CTF Structure and function – not clearly related

8. The GAL4 Protein Yeast activator controls a set of genes responsible for metabolism of galactose The GAL4 protein is a member of the zinc- containing family of DNA-binding proteins

9. Nuclear receptor Zinc module - 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

10. 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

11. 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 - Bind ligands - they activate transcription Type III receptors - ligands are not yet identified

12. 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

13. 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

14. Models for Recruitment

15. 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

16. 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 (homework) –Change in topology –Sliding –Looping –Facilitated tracking

17. Hypotheses of Enhancer Action

18. 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 - by looping out any intervening DNA

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

20. Architectural Transcription Factors Architectural transcription factors are those transcription factors - change the shape control region so that other proteins can interact successfully to stimulate transcription

21. 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

22. 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

23. Homework Explain the four hypotheses of the ability of enhancers to act at a distance. What are insulators? Explain the functions of insulators. Explain mechanism of insulator activity.

24. Study material for exam Structure of activator Three types of DNA binding motif Three types of transcription activation domain What is enhancer? Two types – architectural factors and enhanceosome Recruitment Insulator Ubiquitylation Sumoylation

25. 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

26. Ubiquitylation Ubiquitylation - monoubiquitylation of some activators can have an activating effect Polyubiquitylation marks these same proteins for destruction

27. 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

28. 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

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