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Activators of eukaryotic transcription initiation Proteins which activate transcription initiation by RNA polymerase II and III are called transcription factors. These should not be confused with GTFs that play a more central role in the assembly of the preinitiation complex. Traditional view – transcription factors are sequencespecific DNA-binding proteins. Some recognize upstream promoter elements and influence the transcription only at the promoter to which these elements are attached. Others target sites within enhancers and influence transcription of several genes at once. 1 From T. Brown, Genomes Traditional view: Whether bound to an upstream promoter element or to a more distant enhancer, activator stimulates formation of preinitiation complex (i) by making contact with it, (ii) directly or via another protein, sometimes achieving this (iii) by inducing bend in DNA between the core promoter and the binding site for the TF. 2 From T. Brown, Genomes Repressors of eukaryotic transcription •Repression of initiation is very important in bacteria, but it seemed unlikely that it plays a major control in eukaryotic transcription. •But, a growing number of DNA-binding proteins that repress initiation were found. •They bind at upstream promoter elements or at more distant sites at silencers. •Not really comprehensively studied in eukaryotes. •Some repressors interact with GTFs (TFIIB, TFIID) – affect assembly of preinitiation complex. They may act by inducing chromatin formation over promoter, inducing silencing. 3 From T. Brown, Genomes Control over activators Their activity has to be controlled Activity can be regulated either by controlling the synthesis or by controlling the activity to activate or repress transcription 4 From T. Brown, Genomes Control over the TFs Direct activation – occurs when the extracellular signaling compound is able to enter the cell Indirect activation occurs when the extracellular signaling compound in unable to cross the membrane and instead binds to a cell surface receptor which transduces the signal to the cell interior. 5 Activators - stimulate or inhibit transcription by RNA polymerase II Two functional domains: •DNA-binding domain •Transcription activation domain Also: may have dimerization domain (allows binding of activators to each other) DNA-binding domains DNA binding domains have DNA binding motifs 1. Zinc-containing modules • Zinc-fingers (in TFIIIA or Sp1) • Zinc modules found in the glucocorticoid receptors • Modules containing 2 zinc ions and 6 cysteins, found in GAL4 2. Homeodomains – contain about 60 aa and resemble in structure and function the helix-turn-helix DNA-binding domains.HDs found in many activators, originally were identified in homeobox proteins that regulate development of Drosophila. 3. bZIP and bHLH motifs. The CCAAT/enhancer binding protein, the MyoD protein and many eukaryotic TFs have a highly basic DNA-binding motif linked to one or both of the protein dimerization motifs known as leucine zippers and helix-loop-helix (HLH) motifs. 6 Transcription-activating domains Most activators have one, some have more Main classes: 1. Acidic domains. GAL4 –typical for this group. It has 49 aadomain with 11 acidic aa. 2. Glutamine-rich domains. The activator Sp1 has two such domains, which are about 25% glutamine. One of these has 39 glutamines in a span of 143 aa. Also, Sp1 has two other activating domains that do not fit into any of these main categories. 3. Proline-rich domains. The activator CTF has a domain of 84 aa, 19 of which are prolines. Structures of the DNA-binding motifs of activators Zinc-fingers: •Composed of anti-parallel β-strand, followed by an α-helix. •The β-strand contains two cysteines, and the α-helix two histidines, that are coordinated to a zinc ion. •This coordination of aa to the metal ion helps to form the fingershaped structure. •Specific recognition between the finger and its DNA target occurs in the major groove. 7 Structure of zinc finger 8 9 The GAL4 protein GAL4 – yeast activator that controls set of genes responsible for metabolism of galactose. Each of these GAL4-responsible genes contains a GAL4 target site upstream the transcription start site.These are upstream activator sequences are called UAS. GAL4 binds to UAS as a dimer. •Its DNA-binding motif is located in the first 65aa of the protein. •DNA binding motif of each monomer has 6 cysteines that coordinate two Zn ions. The recognition module contains a short αhelix that protrudes into the DNA major groove and makes a specific interaction there. •Dimetization motif is found in residues 65-94. Nuclear receptors A third class of zinc module is found in nuclear receptors. Nuclear receptors interact with variety of steroid and other hormones. They form hormone-receptor complexes that function as activators by binding to enhancers , or hormone response elements. Thus – they stimulate transcription of associated genes. They have hormone-binding domain. Some exist in cytoplasm complexed with heat-shock protein (hps 90). When hormones are present the receptors dissociate from hps, bind hormone. 10 11 Glucocorticoid receptor action The effect of ligand binding on the receptor is to convert it from a repressing complex to activating complex. 12 Nuclear receptors Other receptors are nuclear (TH receptor). In the absence of hormone they bind to their respective enhancers and repress transcription. In the presence of hormones they form hormone-receptor complexes in the nucleus and function as activators for the same enhancers. Environment determines whether the DNA element to which protein binds, the thyroid response element serves as an enhancer or as a silencer. Homeodomains Homeodomains are DNA-binding domains found in a large family of activators. Their name comes from homeoboxes, that first were discovered in regulatory genes in Drosophila, called homeotic genes. Mutations cause developmental abnormalities. Homeodomains contain DNA-binding motif that functions the same way as prokaryotic helix-turn-helix motifs in which a recognition helix fits into the DNA major groove and makes specific contacts there. N-terminal arm nestles in the adjacent minor groove. 13 The bZIP ans bHLH domains bZIP and bHLH domains combine DNA binding and dimerization. bZIP – leucine zipper and bHLH - helix-loop-helix part They are dimerization domains. Leucine zipper Two alpha helices, leucines interact as zipper 14 Crystal structure of the bZIP motif of GCN4 bound to its target. The bZIP ans bHLH domains Complex between the bHLH domain of MyoD and its DNA target. 15 Independence of the domains of activators DNA binding and transcription-activating domains of activator proteins are independent modules. We can make hybrid proteins with the DNA-binding domain of one protein and the transcription-activation domain of the other one – and the hybrid protein functions as an activator. Functions of activators Recruitment of preinitiation complexes. 16 Interaction among activators Dimerization – great advantage to tge activator, it increases the affinity between activator and its DNA target. Homodimers or heterodimers are formed. The Jun-Fos dimer Jun and fos are proto-oncogenes Jun and Fos proteins are DNA-binding proteins of the bZIP family. Jun dimers are one form of the activator AP1, but Jun and Fos cooperate to bind DNA more tightly than Jun dimers (Fos dimers do not bind at all). Abundant form of AP1 is probably a Jun-Fos heterodimer. bZIP domains are crucial for binding. AP1 target TGACTCA. Multiple enhancers Many genes have more than one enhancer, they respond to multiple stimuli. Examples – metallothionine gene, codes for protein that help to prevent metal poisoning. 17 Architectural transcription factors ATFs change the shape of DNA control region so that other proteins can interact successfully to stimulate transcription. Example – T-cell receptor alpha chain gene control region, which contains three enhancers, binding sites for activators Ets-1, LEF-1 and CREB within just 112 bp of the transcription start site. 18 Tom Maniatis observed similar phenomenon in the human interpheron-β (IFN β) control region, which also has an enhancer close to promoter. Enhancer with its activators Model of enhanceosome assembled at the human IFN β gene. 19 Insulators – block activation of unrelated genes by nearby enhancers – neutral barriers to the influence of neighboring elements How do they work – not really known exactly. Insulators – DNA elements that shield genes from activation or repression by enhancers or silencers. They block such activation or repression on one side of a DNA element, but not on the other. Function: They establish boundaries between DNA regions in a chromosome. No model has been proposed yet as a model of insulator action that can account for all the data available. 20 Mediators Model for activation of a CRE-linked gene. 21 Regulation of transcription factors Activators are subject to a variety of regulation mechanisms. Some are regulated by •Ligand binding •Phosphorylation •Binding to inhibitory proteins •Proteilysis. Signal transduction pathways begin with a signaling molecule that interacts with a receptor on a cell surface, which send a signal inside the cell and frequently leads to altered gene expression. Many signal transduction pathways, including MAP kinase pathway, rely on protein phosphorylation to pass the signal from one protein from another. This amplifies the signal at each step. 22 A signal transduction pathway involving MAP kinase References: 1. R. Weaver, Molecular Biology, 2005 2. B. Lewin, Genes VII, 2000 3. T. Brown, Genomes, 1999. 23