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Lecture PowerPoint to accompany Molecular Biology Fourth Edition Robert F. Weaver Chapter 12 Transcription Activators in Eukaryotes Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 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 12-2 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 12-3 Zinc-Containing Modules • There are at least 3 kinds of zinccontaining modules that act as DNAbinding 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 12-4 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 12-5 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 12-6 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 12-7 12.2 Structures of the DNABinding 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 12-8 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 12-9 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 12-10 The GAL4 Protein • The GAL4 protein is a member of the zinccontaining family of DNA-binding proteins • It does not have a zinc finger • Each GAL4 monomer contains a DNAbinding 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 12-11 on another GAL4 monomer 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 12-12 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 12-13 Glucocorticoid Receptors • DNA-binding domain with 2 zinc-containing modules • One module has most DNA-binding residues • Other module has the surface for proteinprotein interaction to form dimers 12-14 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 12-15 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 12-16 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-loophelix 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 12-17 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 12-18 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 12-19 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 12-20 Models for Recruitment 12-21 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 12-22 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 12-23 Recruitment Model of GAL11Pcontaining 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 12-24 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 12-25 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 12-26 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 12-27 Hypotheses of Enhancer Action 12-28 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 12-29 Control Region of the Metallothionine Gene • Gene product helps eukaryotes cope with heavy metal poisoning • Turned on by several different agents 12-30 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 12-31 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 12-32 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 12-33 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 12-34 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 12-35 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 12-36 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 12-37 Model of Multiple Insulator Action 12-38 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 12-39 Phosphorylation and Activation Replace this area with Figure 12.33: A model for activation of a CRElinked gene 12-40 Activation of a Nuclear Receptor-Activated Gene 12-41 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 12-42 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 12-43 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 12-44 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 12-45 Three Signal Transduction Pathways 12-46 Ras and Raf Signal Transduction 12-47 Wnt Signaling 12-48