Chapter 12 Transcription Activators in Eukaryotes

1. Chapter 12 Transcription Activators in Eukaryotes

2. 12.1 Categories of Activators(激活因子) • Activators (gene-specific TF) can stimulate or inhibit transcription by RNA polymerase II • Structure is composed of at least 2 functional domains (功能域) • DNA-binding domain (DNA结合域) • Transcription-activation domain (转录激活域) • Many also have a dimerization domain (二聚作用域)

3. DNA-Binding Domains • Protein domain (蛋白质结构域)is an independently folded region of a protein • DNA-binding domains have DNA-binding motif (DNA结合基序) • Part of the domain having characteristic shape specialized for specific DNA binding • Most DNA-binding motifs fall into 3 classes 1. Zinc-Containing Modules(含锌组件) 2. Homeodomains(同源域) 3. bZIP and bHLH Motifs(Bzip和bHLH基序)

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 a-helix of the motif into the DNA major groove • Zinc fingers (锌指): TFII A,Sp1 • Zinc modules (锌组件):nuclear receptor • Modules containing 2 zinc and 6 cysteines (含两个锌离子和六个半胱氨酸组件):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 (同源盒蛋白) 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-Activating Domains • Most activators have one of these domains, Some have more than one • These domains fall into three classes: • Acidic domains (酸性域)such as yeast GAL4 with 11 acidic amino acids out of 49 amino acids in the domain • Glutamine-rich domains (谷酰胺富含域) include Sp1 having 2 that are 25% glutamine • Proline-rich domains(脯氨酸富含域) such as CTF which has a domain of 84 amino acids, 19 proline

8. 12.2 Structures of the DNA-Binding Motifs of Activators • DNA-binding domains have well-defined structures • X-ray crystallographic(X射线晶体衍射) studies have shown how these structures interact with their DNA targets • Interaction domains forming dimers (二聚体), or tetramers (四聚体), have also been described • Most classes of DNA-binding proteins can’t bind DNA in monomer form

9. Zinc Fingers • Described by Klug in TFIIIA • Nine repeats of a 30-residue element: • 2 closely spaced cysteines (半胱氨酸) followed 12 amino acids later by 2 closely spaced histidines (组氨酸) • Rich in zinc, enough for 1 zinc ion per repeat • Each zinc ion is complexed by 2 cysteines and 2 histidines in each repeat to form the finger-shaped structure or domain • Specific recognition between the zinc finger and its DNA target occurs in the major groove Zn

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

11. The GAL4 Protein • The GAL4 is a yeast activator that control a set of gene responsible for metabolism of galactose. • Targeting the enhancer: upstream activating sequnces (上游激活序列, UASGS ) • a member of thezinc-containing family • It does not have a zinc finger

12. The GAL4 Protein GAL4 monomer contains a DNA-binding motif with: • 6 cysteines that coordinate 2 zinc ions in a bimetal thiolate cluster (双金属巯基簇) • Short a-helix that protrudes into the DNA major groove is the recognition module • Dimerizationmotif with an a-helix that forms a parallel coiled coil (螺旋圈) as it interacts with the a-helix on another GAL4 monomer

13. The Nuclear Receptors(核受体) • A third class of zinc module is the nuclear receptor • This type of protein interacts with a variety of endocrine-signaling(内分泌信号) 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

14. Type I Nuclear Receptors • Including steroid receptors (内固醇受体)，typified by glucocorticoid receptors [糖(肾上腺)皮质激素] • These receptors reside in the cytoplasm bound to another protein: ligands • When receptors bind to their hormone ligands: • Release their cytoplasmic protein partners • Move to nucleus • Bind to enhancers • Act as activators

15. 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

16. Types II and III Nuclear Receptors • Type II nuclear receptors stay within the nucleus, exemplified by the thyroid hormone (甲状腺激素受体) receptor, • Bound to target DNA sites in both the presence and absence of their ligands. • Without ligands the receptors repress gene activity • When receptors bind ligands, they activate transcription • Type III receptors are “orphan (孤儿)”whose ligands are not yet identified

17. All three classes of zinc-containing DNA-binding modules use a common motif－an -helix－for most of the interaction with their DNA targets.

18. Homeodomains (同源域) • Homeodomains contain three -helices;DNA-binding motif functioning as helix-turn-helix motifs (helix 2 and 3) • A recognition helix(helix 3) fits into the DNA major groove and makes specific contacts there • N-terminal arm nestles in the adjacent minor groove N-terminal arm

19. The bZIP and bHLH Domains The bZIP and bHLH domains combine two functions：DNA binding and dimerization． • The ZIP and HLH parts of the names refer to the leucine zipper(亮氨酸拉链) and helix-loop-helix (螺旋-环螺旋)parts ,respectively; these are the dimerization motifs． • The b in the names refers to a basic region in each domain that forms the majority of the DNA-binding motif． leucine zipper

20. The bZIP and bHLH Domains • To the dimer of bHLH, the basic parts of each long helix to grasp the DNA target site • The leucine zipperputs the adjacent basic regions of each monomer in position to embrace DNA target like a pair of tongs (钳子)

21. 12.3 Independence of the Domains of Activators DNA-binding and transcription-activating domains of activator proteins are independent modules Transcription-activating domain of another (GAL4) with, The hybrid proteins function as an activator DNA-binding domain of one activator (LexA)

22. 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

23. Eukaryotic Activators • Eukaryotic activators also recruit RNA polymerase to promoters • Stimulate binding of general TF 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

24. Models for Recruitment Stepwisebuild-up of preinitiation complex Recruitment of holoenzyme

25. Recruitment of TFIID • Acidic transcription-activating domain of the herpes virus TFVP16 binds to TFIID • TFIID is rate-limiting for transcription in some systems • TFIID is the important target of the VP16 transcription-activating domain

26. Recruitment of the Holoenzyme • Activation in some yeast promoters appears to function by recruitment of holoenzyme A yeast mutant (GAL11P) cause the protein to bind to a region of a dimerization domain of GAL4 GAL11P responded strongly to weak mutant of the activator GAL4. If the holoenzyme is recruited as a unit Any interaction between a activator (bound a promoter) and the holoenzyme recruit the holoenzyme and activate transcription.

27. Recruitment Model of GAL11P-containing Holoenzyme • Dimerization domain of GAL4 binds to GAL11P in the holoenzyme • After dimerization, the holoenzyme, along with TFIID, binds to the promoter, activating the gene

28. 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

29. Dimerization (二聚化作用) • Dimerization is a great advantage to an activator • Dimerization increases the affinity between activator and its DNA target • Some activators form homodimers (同型二聚体) • Heterodimers(异型二聚体) are also formed • Products of the jun and fos genes form a heterodimer

30. Action at a Distance (远距离效应) • 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

31. Hypotheses of Enhancer Action (a)changes in topologyopens the promoter to general TF Activator binds to an enhancer (b) slides the promoter  activate transcription (C) looping out the DNA between E and P binds to general TF/pol and facilitates general TF and pol binding to promoter (d) Facilitated tracking, looping out short DNA segment, repeat then same (C)

32. Complex Enhancers • Many genes can have more than one activator-binding site permitting them to respond to multiple stimuli (刺激) • Control Region of the Metallothionine (金属硫蛋白) Gene • Gene product helps eukaryotes cope with heavy metal poisoning • Turned on by several different agents

33. Architectural Transcription Factors结构转录因子 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

34. An Architectural Transcription Factor Example • human T-cell receptor -chain(TCRa)gene • Within 112 bp upstream of the start of transcription are 3 enhancer elements • These elements bind to:Ets-1, LEF-1(Lymphoid Enhancer-binding Factor), CREB • LEF-1 binds to the minor groove of its DNA target through its HMG (High Mobility Group)domain and induces strong bending of DNA • DNA Bending Aids Protein Binding(activators and general TF) • LEF-1 does not enhance transcription by itself

35. Enhanceosome(增强体) • 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

36. Insulator(绝缘子) DNA elements to block activation (or Barrier activity) of unrelated genes by nearby enhancers (silencer)． Barrier activity (屏障活性): insulator between promoter and condensed, repressive chromatin prevents promoter from being repressed Enhancer-blocking activity(增强子屏蔽活性): insulator between promoter and enhancer prevents the promoter from being activated

37. Mechanism of Insulator Activity Sliding model:Activator bound to an enhancer and stimulator slides along DNA from enhancer to promoter, and the insulator blocks the progression of this signal． Looping model:Two insulators flank an enhancer, when bound they interact with each other isolating enhancer

38. 12.6 Regulation of Transcription Factors • Phosphorylation (磷酸化) of activators (辅助激活因子) can allow them to interact with coactivators that in turn stimulate transcription • Ubiquitylation (泛素化) of transcription factors can mark them for • Destruction by proteolysis (蛋白质水解) • Stimulation of activity • Sumoylation (SUMO修饰) is the attachment of the polypeptide SUMO which can target for incorporation into compartments of the nucleus • Methylation (甲基化) and acetylation(乙酰化)can modulate activity