Bài giảng Molecular Biology - Chapter 12 Transcription Activators in Eukaryotes

Molecular BiologyFifth EditionChapter 12Transcription Activators in EukaryotesLecture PowerPoint to accompanyRobert F. WeaverCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.1Transcription Activators of EukaryotesThe general transcription factors by themselves dictate the starting point and direction of transcription but they are capable of sponsoring only a low level of transcription or basal transcriptionTranscription of active genes in cells rises above the basal levelEukaryotic cells have additional, gene-specific transcription factors called activators that bind to DNA elements called enhancers to provide the extra needed boost to transcription212.1 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 domain3DNA-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 classes; zinc-containing modules, homeodomains and bZIP and bHLH motifs4Zinc-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 cysteines5HomeodomainsThese 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 development6bZIP 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 protein7Transcription-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 proline8SummaryEukaryotic activators are composed of at least two domains: a DNA-binding domain and a transcription-activating domainDNA-binding domains contain motifs such as zinc modules, homeodomains, and bZIP or bHLH motifsTranscription activating domains can be acidic, glutamine-rich or proline-rich912.2 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 form10Zinc FingersDescribed by Klug in GTF 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 groove11Arrangement of Three Zinc Fingers in a Curved ShapeThe zinc finger is composed of: An antiparallel b-strand that contains 2 cysteines2 histidines in an a-helixHelix and strand are coordinated to a zinc ion12The GAL4 ProteinThe GAL4 protein is a member of the zinc-containing family of DNA-binding proteinsEach 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 monomer13The 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 genes14Type 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 activators15Glucocorticoid ReceptorsDNA-binding domain with 2 zinc-containing modulesOne module has most DNA-binding residuesOther module has the surface for protein-protein interaction to form dimers16Types II and III Nuclear ReceptorsType II nuclear receptors stay within the nucleus bound 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 identified17Homeodomain-DNA ComplexHomeodomains 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 groove18The 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 zippers1912.3 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 anotherThe hybrid protein still functions as an activator2012.4 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 recruitment21Eukaryotic 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 holoenzyme22Models for Recruitment of Preinitiation Complex Components in Yeast23Recruitment 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 24Recruitment 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 common25Recruitment 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 gene2612.5 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 gene27DimerizationDimerization is a great advantage to an activator as it increases the affinity between the activator and its DNA targetSome activators form homodimers but others function as heterodimers28Action 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 tracking29Hypotheses of Enhancer Action303C: Method to detect DNA loopingChromosome conformation capture (3C) is a technique used to determine if enhancer action requires DNA loopingUsed to test whether two remote DNA regions, such as an enhancer and a promoter, are brought together 31Genomic ImprintingBecause most eukaryotes are diploid organisms, you would predict that it does not matter which allele of any given gene came form the mother or the fatherThis is true in most cases but there are important exceptionsThe differences between the genes resides in how they are modified, or imprinted, differently in females and malesEvidence exists in mice and humans32Transcription FactoriesDiscrete nuclear sites where transcription of multiple genes occursIf two or more genes on the same chromosome are clustered in the same transcription factory, DNA loops would naturally form between themThus, the existence of transcription factories implies the existence of DNA loops in eukaryotic cells33Evidence for Transcription FactoriesCook and colleagues counted the number of transcription factories by labeling growing RNA chains in HeLa cells with BrU followed by permeabilization and further labeling with biotin-CTP detected with anti-BrU or anti-biotin primary antibodies followed by secondary antibodies labeled with gold particlesThey concluded that transcription is associated with the clusters, not the single particles34Complex 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 DNA35Control Region of the Metallothionine GeneThe metallothionine gene product helps eukaryotes cope with heavy metal poisoningTurned on by several different agentsComplex enhancers enable a gene to respond differently to different combinations of activatorsThis gives cells exquisitely fine control over their genes in different tissues, or at different times in a developing organism36Architectural 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 transcription37Example of Architectural Transcription Factor: Control region of the human TCR  chain geneWithin 112 bp upstream of the start of transcription are 3 enhancer elementsThese elements bind to:Ets-1, LEF-1, CREB38EnhanceosomeAn enhanceosome is a nucleoprotein complex containing a collection of activators bound to an enhancer in such a way that stimulates transcriptionThe archetypal enhanceosome involves the IFN enhancer with a structure that involves eight polypeptides bound cooperatively to an essentially straight 55-bp strech of DNA39DNA 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 factors40InsulatorsInsulators can shield genes from activation by enhancers (enhancer blocking activity)Insulators can shield genes from repression by silencers (barrier activity)41Mechanism 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 enhancer42Model of Multiple Insulator Action43SummarySome insulators have both enhancer-blocking and barrier activities, but some have only one or the otherInsulators may do their job by working in pairs that bind proteins that can interact to form DNA loops that would isolate enhancers and silencers so they can no longer stimulate or repress promotersInsulators may establish boundaries between DNA regions in a chromosome4412.6 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 activity45Phosphorylation and Activation: A Model for activation of a CRE-linked geneReplace this area with Figure 12.33: A model for activation of a CRE-linked gene46Model for the Activation of a Nuclear Receptor-Activated Gene47UbiquitylationUbiquitylation, 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 transcription48Activator 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 stable49Activator 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 effects50Signal 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 step51Three pathways that use CBP/p300 to mediate transcription activation52Ras and Raf Signal Transduction53