Molecular biology - Chapter 12: Transcription activators in eukaryotes

Molecular BiologyFourth EditionChapter 12Transcription Activators in EukaryotesLecture PowerPoint to accompanyRobert F. WeaverCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.112.1 Categories of ActivatorsActivators can stimulate or inhibit transcription by RNA polymerase IIStructure is composed of at least 2 functional domainsDNA-binding domainTranscription-activation domainMany also have a dimerization domain2DNA-Binding DomainsProtein domain is an independently folded region of a proteinDNA-binding domains have DNA-binding motifPart of the domain having characteristic shape specialized for specific DNA bindingMost DNA-binding motifs fall into 3 classes3Zinc-Containing ModulesThere are at least 3 kinds of zinc-containing modules that act as DNA-binding motifsAll use one or more zinc ions to create a shape to fit an a-helix of the motif into the DNA major grooveZinc fingersZinc modulesModules containing 2 zinc and 6 cysteines4HomeodomainsThese domains contain about 60 amino acidsResemble the helix-turn-helix proteins in structure and functionFound in a variety of activatorsOriginally identified in homeobox proteins regulating fruit fly development5bZIP and bHLH MotifsA number of transcription factors have a highly basic DNA-binding motif linked to protein dimerization motifsLeucine zippersHelix-loop-helixExamples include:CCAAT/enhancer-binding proteinMyoD protein6Transcription-Activating DomainsMost activators have one of these domainsSome have more than oneAcidic domains such as yeast GAL4 with 11 acidic amino acids out of 49 amino acids in the domainGlutamine-rich domains include Sp1 having 2 that are 25% glutamineProline-rich domains such as CTF which has a domain of 84 amino acids, 19 proline712.2 Structures of the DNA-Binding Motifs of ActivatorsDNA-binding domains have well-defined structuresX-ray crystallographic studies have shown how these structures interact with their DNA targetsInteraction domains forming dimers, or tetramers, have also been describedMost classes of DNA-binding proteins can’t bind DNA in monomer form8Zinc FingersDescribed by Klug in TFIIIANine repeats of a 30-residue element:2 closely spaced cysteines followed 12 amino acids later by 2 closely spaced histidinesCoordination of amino acids to the metal helps form the finger-shaped structureRich in zinc, enough for 1 zinc ion per repeatSpecific recognition between the zinc finger and its DNA target occurs in the major groove9Arrangement of Three Zinc Fingers in a Curved ShapeThe zinc finger is composed of: An antiparallel b-strand contains the 2 cysteines2 histidines in an a-helixHelix and strand are coordinated to a zinc ion10The GAL4 ProteinThe GAL4 protein is a member of the zinc-containing family of DNA-binding proteinsIt does not have a zinc fingerEach GAL4 monomer contains a DNA-binding motif with:6 cysteines that coordinate 2 zinc ions in a bimetal thiolate clusterShort a-helix that protrudes into the DNA major groove is the recognition moduleDimerization motif with an a-helix that forms a parallel coiled coil as it interacts with the a-helix on another GAL4 monomer11The Nuclear ReceptorsA third class of zinc module is the nuclear receptorThis type of protein interacts with a variety of endocrine-signaling moleculesProtein plus endocrine molecule forms a complex that functions as an activator by binding to hormone response elements and stimulating transcription of associated genes12Type I Nuclear ReceptorsThese receptors reside in the cytoplasm bound to another proteinWhen receptors bind to their hormone ligands:Release their cytoplasmic protein partnersMove to nucleusBind to enhancersAct as activators13Glucocorticoid ReceptorsDNA-binding domain with 2 zinc-containing modulesOne module has most DNA-binding residuesOther module has the surface for protein-protein interaction to form dimers14Types II and III Nuclear ReceptorsType II nuclear receptors stay within the nucleusBound to target DNA sitesWithout ligands the receptors repress gene activityWhen receptors bind ligands, they activate transcriptionType III receptors are “orphan” whose ligands are not yet identified15HomeodomainsHomeodomains contain DNA-binding motif functioning as helix-turn-helix motifsA recognition helix fits into the DNA major groove and makes specific contacts thereN-terminal arm nestles in the adjacent minor groove16The bZIP and bHLH DomainsbZIP proteins dimerize through a leucine zipperThis puts the adjacent basic regions of each monomer in position to embrace DNA target like a pair of tongsbHLH proteins dimerize through a helix-loop-helix motifAllows basic parts of each long helix to grasp the DNA target sitebHLH and bHLH-ZIP domains bind to DNA in the same way, later have extra dimerization potential due to their leucine zippers1712.3 Independence of the Domains of ActivatorsDNA-binding and transcription-activating domains of activator proteins are independent modulesMaking hybrid proteins with DNA-binding domain of one protein, transcription-activating domain of anotherSee that the hybrid protein still functions as an activator1812.4 Functions of ActivatorsBacterial core RNA polymerase is incapable of initiating meaningful transcriptionRNA polymerase holoenzyme can catalyze basal level transcriptionOften insufficient at weak promotersCells have activators to boost basal transcription to higher level in a process called recruitment19Eukaryotic ActivatorsEukaryotic activators also recruit RNA polymerase to promotersStimulate binding of general transcription factors and RNA polymerase to a promoter2 hypotheses for recruitment:General TF cause a stepwise build-up of preinitiation complexGeneral TF and other proteins are already bound to polymerase in a complex called RNA polymerase holoenzyme20Models for Recruitment21Recruitment of TFIIDAcidic transcription-activating domain of the herpes virus transcription factor VP16 binds to TFIID under affinity chromatography conditionsTFIID is rate-limiting for transcription in some systemsTFIID is the important target of the VP16 transcription-activating domain 22Recruitment of the HoloenzymeActivation in some yeast promoters appears to function by recruitment of holoenzymeThis is an alternative to the recruitment of individual components of the holoenzyme one at a timeSome evidence suggests that recruitment of the holoenzyme as a unit is not common23Recruitment Model of GAL11P-containing HoloenzymeDimerization domain of FAL4 binds to GAL11P in the holoenzymeAfter dimerization, the holoenzyme, along with TFIID, binds to the promoter, activating the gene2412.5 Interaction Among ActivatorsGeneral transcription factors must interact to form the preinitiation complexActivators and general transcription factors also interactActivators usually interact with one another in activating a geneIndividual factors interact to form a protein dimer facilitating binding to a single DNA target siteSpecific factors bound to different DNA target sites can collaborate in activating a gene25DimerizationDimerization is a great advantage to an activatorDimerization increases the affinity between activator and its DNA targetSome activators form homodimersHeterodimers are also formedProducts of the jun and fos genes form a heterodimer26Action at a DistanceBacterial and eukaryotic enhancers stimulate transcription even though located some distance from their promotersFour hypotheses attempt to explain the ability of enhancers to act at a distanceChange in topologySliding Looping Facilitated tracking27Hypotheses of Enhancer Action28Complex EnhancersMany genes can have more than one activator-binding site permitting them to respond to multiple stimuliEach 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 DNA29Control Region of the Metallothionine GeneGene product helps eukaryotes cope with heavy metal poisoningTurned on by several different agents30Architectural 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 transcription31An Architectural Transcription Factor ExampleWithin 112 bp upstream of the start of transcription are 3 enhancer elementsThese elements bind to:Ets-1LEF-1CREB32EnhanceosomeAn enhanceosome is a complex of enhancer DNA with activators contacting this DNAAn example is the HMG that helps to bend DNA so that it may interact with other proteins33DNA Bending Aids Protein BindingThe activator LEF-1 binds to the minor groove of its DNA target through its HMG domain and induces strong bending of DNALEF-1 does not enhance transcription by itselfBending it induces helps other activators bind and interact with activators and general transcription factors34Examples of Architectural Transcription FactorsBesides LEF-1, HMG I(Y) plays a similar role in the human interferon-b control geneFor the IFN-b enhancer, activation seems to require cooperative binding of several activators, including HMG I(Y) to form an enhanceosome with a specific shape35InsulatorsInsulators act by:Enhancer-blocking activity: insulator between promoter and enhancer prevents the promoter from being activatedBarrier activity: insulator between promoter and condensed, repressive chromatin prevents promoter from being repressed36Mechanism of Insulator ActivitySliding modelActivator bound to an enhancer and stimulator slides along DNA from enhancer to promoterLooping modelTwo insulators flank an enhancer, when bound they interact with each other isolating enhancer37Model of Multiple Insulator Action3812.6 Regulation of Transcription FactorsPhosphorylation of activators can allow them to interact with coactivators that in turn stimulate transcriptionUbiquitylation of transcription factors can mark them for Destruction by proteolysisStimulation of activitySumoylation is the attachment of the polypeptide SUMO which can target for incorporation into compartments of the nucleusMethylation and acetylation can modulate activity39Phosphorylation and ActivationReplace this area with Figure 12.33: A model for activation of a CRE-linked gene40Activation of a Nuclear Receptor-Activated Gene41UbiquitylationUbiquitylation, especially monoubiquitylation, of some activators can have an activating effectPolyubiquitylation marks these same proteins for destructionProteins from the 19S regulatory particle of the proteasome can stimulate transcription42Activator SumoylationSumoylation is the addition of one or more copies of the 101-amino acid polypeptide SUMO (Small Ubiquitin-Related Modifier) to lysine residues on a proteinProcess is similar to ubiquitylationResults quite different – sumoylated activators are targeted to a specific nuclear compartment that keeps them stable43Activator AcetylationNonhistone activators and repressors can be acetylated by HATsHAT is the enzyme histone acetyltransferase which can act on nonhistone activators and repressorsSuch acetylation can have either positive or negative effects44Signal Transduction PathwaysSignal transduction pathways begin with a signaling molecule interacting with a receptor on the cell surfaceThis interaction sends the signal into the cell and frequently leads to altered gene expressionMany signal transduction pathways rely on protein phosphorylation to pass the signal from one protein to anotherThis leads to signal amplification at each step45Three Signal Transduction Pathways46Ras and Raf Signal Transduction47Wnt Signaling48