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Chapter 12

BIOL 367 Chapter 12: biol 367 chapter 12

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BIOL 367
Reg Storms

CHAPTER 12 – TRANSCIPTION ACTIVATORS IN EUKARYOTES Transcription Activators (gene specific transcription factors) and enhancers (the binding sites for gene specific transcription factors) in eukaryotes - Heterochromatin = highly condensed and inaccessible to RNA polymerases, so it cannot be transcribed - Euchromatin = still contains protein, but is relatively extended Categories of Activators - Activators can either stimulate or inhibit transcription by RNA polymerase II - They have structures composed of at least 2 functional domains: a DNA binding domain and a transcription- activating domain - Many also have a dimerization domain that allows the activators to bind to each other (homodimers, heterodimers, higher multimers) - Some even have binding sites for effector molecules (steroid hormones) DNA-Binding Domains - Each DNA-binding domain has a DNA-binding motif, which is the part of the domain that has a characteristic shape specialized for specific DNA binding - Most classes: 1. Zinc-containing modules all use one or more zinc ions to create the proper shape so an alpha helix within the motif can fit into the DNA major groove and make specific contacts there a) Zinc Ringers (ex. TFIIIA, Sp1) b) Zinc modules found in the glucocorticoid receptor and other members of this group of nuclear receptors c) Modules containing two zinc ions and six cysteines, found in the yeast activator GAL4 and its relatives 2. Homeodomains resemble in structure and function the helix-turn-helix DNA-binding domains 3. bZIP and bHLH motifs 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 1 Transcription-Activating Domains - most activators have one of these domains, but some have more than one 1. acidic domains – ex. yeast activator Gal4 2. glutamine-rich domains –ex Sp1 has two 3. proline rich domains – ex. CTF - the acidic activation domain of GAL4 tends to form a defined structure – a beta sheet – in slightly acidic solution Structures of the DNA-Binding Motifs of Activators - most classes of DNA-binding proteins are incapable of binding to DNA in monomer form; they must at least from dimers to function Zinc Fingers - this finger shape by itself does not confer any binding specificity, since there are many different finger proteins, all with the same shape fingers but each binding to its own unique DNA target sequence. Thus, it is the precise amino acid sequence Interaction with DNA - all three Zif268 fingers lining up in the major groove, arranged in a curve, or C shape, which matches the curve of the DNA double helix Comparison with Other DNA-binding proteins - beta sheet in Zif268 seems to serve the same fuction as the first alpha helix in a helix-turn-helix protein,to bind to the DNA bcakbone and help position the recognition helix for optimal ineractionwith the DNA major groove - these proteins do not need to form dimers or tetramers to bind to DNA. They already have multiple binding domains built in. - most of the protein-DNA contacts are with one DNA strand, rather than both - most of the contacts are with abses, rather than the DNA backbone The GAL4 Protein - the GAL4 protein is a yeast activator that contorls a set of genes responsible for metabolism of galactose. Each of these GAL4-responsive genes contains a GAL4 target site. These target sites are called upstream activaitng sequences, or UAS s. G - GAL4 binds to UAS asGa dimer 2 The DNA binding motif - One end of each monomer contains a DNA binding motif contianing 6cysteines that complex 2 zinc ions, forming a bimetal thiolate cluster. Each of these motifs also features a short alpha helix that protrudes into the major groove of the DNA double helix, where its amino acid side chains can make specific interactions wit hteh DNA bases and backbone. The other end of each monomer is al alpha helix that serves a dimerization function The dimerization motif - Alpha helices form a parallel coiled coil - The dimerizing alpha helices point directly at the minor groove of the DNA The Nuclear Receptors - Nuclear receptors interact with a variety of endocrine signaling molecules (steroids and other hormones) - They form hormone receptor complexes that funciton as acivators by binding to enhancers, or hormone response elements,and stimulating transcription of their associated genes 3 classes: - Type I receptors include the steroid hormone receptors, typified by the glucocorticoid receptor. In the absence of their hormone ligands, these receptors reside in the cytoplasm, cupled with another protein. When a type I receptor binds to its hormone ligand, itreleases its protein partner and migrates to the nucleus, where it binds as a homodimer to its hormone response element o Aspects of protein-DNA interaction: (1) the binding domain dimerizes, with each monomer making specific contacts with one target half site. (2) Each binding motif is a zinc moleculethat contains two zinc ions,rather than the one found inclassic zinc fingers. (3) each zinc ion is complexed tofour cysteines toform a finger-like shape. (4) the amino terminal finger in eachbinding domain enganges in most of the ineractions with the DNA target. Most of the interactions involve an alpha-helix - The type II receptors, exemplified by the thyroid hormone receptor, stay in the nucleus, where they form dimers with another protein called retinoic acid receptor x (RXR) whose ligand is 9-cis-retinoic acid. These receptors bind to their target sites in both the presence and absence of their ligands. o Binding of these type II receptors in the absence of ligand can reprses transcription, whereasbinding of the receptors along with their ligands can stimulate transcription - Type III receptors are not as well understood. Known as orphan receptors because their ligands have not been identified 3 Homeodomains - The DNA binding domains found in a large family of activators - Homeoboxes - Members of the helix-turn-helix family - Each homeodomain contains 3 alpha helices; the second and third of these form the h elix-turn-helix motif,with the third serving as the recognition helix - The N-terminus of the protein forms an arm that insertss into theminor groove of the DNA - Most homeodomain proteins have weak DNA-binding specificity on their own. The bZIP and bHLH Domains - The bZIP and bHLH domains combine two functions: DNA binding and dimerization - The ZIP and HLH pasrts = leucine zipper and helix-loop-helix - B = basic region - bZIP domain consists of two polypeptides, eac of which contains half of the zipper: an alpha helix with leucine (or other hydrophobic) residues spaced seven amino acids apart, so they are all on one face of the helix  similar string of amino acids on the otherp rotein monomer = two halves of a zipper - bHLH domain = the helix-loop-helix part is the idmerization motif, but the long helix (helix 1) in each HLH domain contains the basic region of the domain, which grips the DNA target via its major groove, just as the bZIP domain does. 4 - in the absence of galactose, gal4 binds tothe UAS (upstream activator sequence) and this UAS is only found upstream of galactose utilization genes. - absence - gal 80 binds to gal 4, hides the activator domains - in the presence of galactose, galactose binds to gal3, gal3 gets converted to another conformation and binds to gal 80 w/ higher affinity than gal 80 binds to gal 4 - now it can facilitate the general transcription factors and shit to the core promoter - transcription activation of gal genes involves the addition of galactose to the media, it binds ot gal3, diff conformation enables gal 3 to bind to gal 80 and then it sequesters it so that there’s insufficient gal8- to bind to gal4 - gal 80 = transcription repressor Take Home: The distinctly different expression pattern of the yeast GAL1 and CYC1 genes is determined by each gene’s enhancer /UAS region Enhancer regions are essential for gene expression 5 Independence of the Domains of Activators - Scientists demonstrated the independence of the domains by creating a chimeric factor with the DNA-binding domain o fone protein and the transcription-activating domain of the other. This hybrid protein funcitoned as an activator, with its specificity dictated by its DNA-binding domain - LexA is a prokaryotic repressor that binds to lexA operators and reprseses downstream genes in E. coli cells - A chimeric gene containing the coding regions for the transcription-activating omain of GAL4 and the DNA-protein product of this gene, they introduced two plasmdis into yeast cells. - The first plasmid had the chimeric gene, which produced its hybrid product. - Thes second contained a promoter responsive to GAL4 (either the GAL1 or the CYC1 promoter), linked to the E. coli beta-galactosidase gene, which served as a reporter gene - To make the GAL1 promoter responsive ot activation, the investigators had to itnroduce a DNA target for the LeA DN-binding domain. - Did the chimeric protein activate the GAL1? Yes - When no DNA target site was present, no beta galactosidase oculd be made. - When the lexA operator replaced UAS , the LGxA-Gal4 chimeric protein could activate the beta- galactosidase production over 500-fold. - Thus, one can replace the DNA-binding domain of GAL4 with the DNA-binding domain of a completely unreleated protein, and produce a functionall activator. 6 Functions of Acviators - In bacteria, the core RNA polymerase is incapapble of initiation meaningful transcription, but the RNA polymerase holoenzyme can catalyze basal level transcription. - Cells need activators to boost to higher levels by recruitment - Recruitment leads to tight binding of RNA polymerase holoenzyme to a promoter - Eukarytoic activators als orecruit RNA pol to promoters, but not as directly as prokaryotic activators - The eukaryotic activators stimulate binding of general transcription factors and RNA pol to a promoter - Two hypotheses (1) the general transcirption factors cause a stepwise build- up of a preinitiation complex, or (2) the general transcription factors and other proteins are already bound to the polymerase in a complex called the RNA polymerase II holoenzyme, - Truth may be a combo of the two - Direct contacts between general transcription factors and activators are necessary for recruitment Recruitment of TFIID - Experiments to identify the factor that binds to the acidic transcription-activating domain of the herpes virus transcription factor VP16 - To find out what proteins bind to the VP16 activating domain, they poured HeLa cell nuclear extracts through the columns containing either protein A by itself or the protein A/VP16- activating domain fusion protein - Then they used run off transcription - TFIID is the important target of the VP16 transcription activating domain in this experimental system 7 Recruitment of the Holoenzyme Evidence for recruitment of the holoenzyme as a unit - If the holoenzyem is recrutied as a unit, then interaction between any part of the activator (bound neaer a promoter) and any part of the holoenzyme should serve to recruit the holoenzyme to the promoter - On the other hand, if the preinitiation complex must be built up protein by protein, then an abnormal itneractino between an activator and a seemingly unimportant member of the holoenzyme should not activate transcription - Yeast mutant with a point mutation that changed a single amino acid in a holoenzyme protein (GAL11). - Named altered protein GAL11P (potentiator) because it responded strongly ot weak mutatn versions of the activator GAL4 - The alteration in GAL11 caused this protein tobind to a region of the dimerization domain of GAL4 - Because GAL11 is aprt of the holoenzyme, this novel association between GAL11P and GAL4 should recruit the holoenzyme to GAL4-responsiv
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