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Ch 12. Transcription Activators in Eukaryotes Gene-specific transcription factor Transcriptional Control • General transcription factor ; • starting point • direction of transcription • basal level transcription • Activator ; • trans-acting element ; gene regulatory protein • cis-acting element ; regulatory DNA sequence /enhancer • gene-specific transcription • extra boost in transcription • regulate chromatin structure & Gene activity Protein folding & Motif Transcription Activators • Functional domains • DNA-binding domain (motif) – Zinc-containing modules ; Zinc finger (Sp1, TFIIIA), GR, GAL4 – Homeodomain (HD); l phage repressor, HTH – bZIP & bHLH; C/EBP, MyoD, Leu Zipper, HLH • Transcription-activating domain – acidic domains; GAL4 – glutamaine-rich domain; Sp1 – proline-rich domain; CTF • Others, – dimerization domain (Pt-Pt interaction), – effector (ligand) binding domain (in steroid hormone receptors) Zinc fingers: C2H2 finger, Y/F-C-X2-4-C-X3-F/Y-X5-L-X2-H-X3-4-H -helix + anti-parallel -ribbon + Zn two C in -ribbon, two H in -helix, basic a.a. in -helix TFIIIA 9 repeats of 30-residues NMR study of Xfin finger Multiple fingers interact with major groove of target DNA X-ray crystallography of Zif268 a.a. in -helix directly interact with target sequence, -strand interacts with DNA backbone & help proper positioning of -helix Gal4 protein: C6 Zn motif, C-X2-C-X6-C-X5-6-C-X2-C-X6-C interact with two Zn (1:3) dimerization by forming coiled coil, Dimerization helix at minor groove Recognition helix at major groove DNA recognition motif of GAL4 Zinc modules in nuclear receptors Hormone receptors; androgen, estrogen, progesterone, glucocorticoid, vitamin D, thyroid hormone, retinoic acid Three domains; - DNA binding - Activation - Hormone binding Type I : glucocorticoid Rc Type II : thyroid Rc & RXR (in nucleus) activator vs. repressor Type III : orphan receptor hsp90 C4 Zn finger: C-X2-C-X13-C-X2-C-X14-15-C-X5-C-X9-C-X2-C two fingers, each C4 finger binds a Zn ion need dimerization for binding Homeodomain Antennapedia bithorax Homeodomains: helix-turn-helix motif • three -helix, one(#3) for major groove interaction • weak DNA binding specificity; need other protein for specific & efficient binding N-terminal arm into the minor groove bZIP & bHLH Leucine Zipper: leucines in -helix with 7 a.a. interval ZIP of GCN4: parallel coiled coil bHLH Independence of the domains of activators Hybrid of yeast GAL4(AD) + bacterial LexA(BD) Functions of Activators Recruitment of General Transcription Factors Recruitment of RNA polymerase Two models of recruitment of pre-initiation complex; stepwise vs. holoenzyme Functions of Activators; recruitment of TFIID Affinity column with protein A-VP16 activation domain HeLa cell extract eluate In vitro transcription VP16-bound factor recovers transcription; TFIID Recruitment of holoenzyme Recruitment of Holoenzyme GAL11; a component of holoenzyme GAL11p mutant – potentiates response to GAL4 activator GAL11p binds to GAL4 through interaction with dimerization domain GAL11-LexA fusion can activate transcription, confirming holoenzyme recruitment model Interaction among activators: dimerization Increase affinity, specificity, additional regulation Jun, Fos: bZIP family Dimerization; Jun+Fos= AP1 TRE (TPA response element) Jun homodimer - weak binding Jun-Fos - strong binding Fos dimer - no binding Action at a distance Looping effect can be mimicked by catenane formation Catenane experiments Catenated link between enhancer and test promoter (Ψ40) allowed activation Multiple enhancers Glucocorticoid RE basal level enhancer metal RE human metallothionine gene Modular arrangement of enhancers at sea urchin Endo16 gene Interaction between enhancers Organized regulations of six modules of many elements during development Different responses of enhancers in different environments Combination of enhancer modules linked to CAT reporter tested in sea urchin development Architectural Transcription Factors Looping is difficult – too close to the promoter LEF-1 (Lymphoid enhancer binding factor) no activation by itself, only help activation by other factors HMG domain; binds to minor groove & bends DNA Transcription factors bind close together hTCRα Model of Enhanceosome Insulators DNA elements to block activation or repression from nearby elements GAGA boxes & binding protein, Trl in Drosophila, Enhancer blocking Barrier Mechanism of insulator activity ; Two hypothesis Sliding model Looping model Multiple insulator action Effect of insulators on gene expression Activators (repressors) Transcriptional Regulators Transcription machinery Two classes of global regulators Regulators affecting chromatin structure: SWI/SNF & ISWI chromatin-remodeling complexes, histone acetyltransferase (HAT) complexes histone deacetyltransferase (HDAC) complexes Regulators acting through RNA pol & its associated proteins mediator complexes in yeast & mammalian cells Regulation of transcriptional factors • Nuclear receptors / ligands ; translocate to the nucleus and activate Tc • Conformational change from repressor to activator • Phosphorylation for the interaction with co-activators • Ubiquitination : proteolysis vs. Stimulation • Sumoylation • Methylation • Acetylation Co-activator of transcription regulation DNA + GTFs + RNA pol + Activators no activation : need something else (mediators) Activator interference: Squelching • increase of one activator inhibits another activator • addition of GTFs no effects • activators compete for common limiting factor Purification of mediator in yeast system (by Kornberg) SMCC/TRAP, CRSP in mammal Signal transduction by PKA Co-activator ; CBP/p300 Steroid receptor coactivator (SRC) Multiple roles of CBP/P300 Protein degradation Signaling Molecules and Cell surface Receptors • Endocrine ; Hormones (blood) • Paracrine ; neurotransmitter, growth factors (diffusion) • Autocrine ; growth factors (tumor) • Integral membrane protein Mechanisms of signal transduction • Changes in the activity of pre-existing proteins • Changes in the amounts of protein via gene transcription 7 major classes of cell surface receptors Signal transduction pathway involving Ras/Raf G protein-coupled receptors (GPCR) • Seven membrane-spanning regions • Cytosolic segments are involved in interaction with coupled trimeric G proteins • G ; GTPase switch protein activate or inhibit effector protein (Adenylyl Cyclase) Activation of phospholipase C by GPCR • Phosphatidiylinositol (PI) ; phosphorylated by PI kinase • Phosphoinositide (PIP, PIP2) ; cleaved by PLC to DAG and IP3 • IP3 triggers release of Ca2+ from ER • DAG activates protein kinase C (PKC) • Ca2+ /calmodulin complex ; transcriptional regulation Intracellular signal transduction : General principles • Second messengers carry signals from many receptors ; intracellular concentration • Conserved intracellular proteins function in signal transduction ; G protein, Kinase & Phosphatase • Localization of receptors ; protein-protein, protein-lipid • Regulation of signaling pathways & Fine-tuning of cellular activity 2nd messenger Short-lived increase/decrease in the concentration of certain low-molecular weight Intracelluar signaling molecules Ca2+ Signal Transduction Cascade Regulation of signaling pathways 1. Degradation of second messenger 2. Deactivation of signal transduction protein 3. Desensitization of receptors at high signal concentration or prolonged exposure to a signal • Endocytosis of receptors • modifying the binding activity to ligand • phosphorylation of receptors or binding of inhibitory protein Interaction of different signaling pathways ; fine-tuning of cellular activities