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Published byEvangeline Mavis Merritt Modified over 8 years ago
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Ch 12. Transcription Activators in Eukaryotes Gene-specific transcription factor
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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
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Protein folding & Motif
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Transcription Activators Functional domains DNA-binding domain (motif) –Zinc-containing modules ; Zinc finger (Sp1, TFIIIA), GR, GAL4 –Homeodomain (HD); 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)
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Zinc fingers: C2H2 finger, Y/F-C-X 2-4 -C-X 3 -F/Y-X 5 -L-X 2 -H-X 3-4 -H -helix + anti-parallel -ribbon + Zn two C in -ribbon, two H in -helix, basic a.a. in -helix NMR study of Xfin finger TFIIIA 9 repeats of 30-residues
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Multiple fingers interact with major groove of target DNA a.a. in -helix directly interact with target sequence, -strand interacts with DNA backbone & help proper positioning of -helix X-ray crystallography of Zif268
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Gal4 protein : C6 Zn motif, C-X 2 -C-X 6 -C-X 5-6 -C-X 2 -C-X 6 -C interact with two Zn (1:3) dimerization by forming coiled coil, Dimerization helix at minor groove Recognition helix at major groove
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DNA recognition motif of GAL4
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Zinc modules in nuclear receptors Hormone receptors; androgen, estrogen, progesterone, glucocorticoid, vitamin D, thyroid hormone, retinoic acid Three domains; - DNA binding - Activation - Hormone binding hsp90 Type I : glucocorticoid Rc Type II : thyroid Rc & RXR (in nucleus) activator vs. repressor Type III : orphan receptor
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C4 Zn finger: C-X 2 -C-X 13 -C-X 2 -C-X 14-15 -C-X 5 -C-X 9 -C-X 2 -C two fingers, each C4 finger binds a Zn ion need dimerization for binding
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Homeodomain Antennapedia bithorax
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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
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bZIP & bHLH Leucine Zipper: leucines in -helix with 7 a.a. interval
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ZIP of GCN4: parallel coiled coil
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bHLH
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Independence of the domains of activators Hybrid of yeast GAL4(AD) + bacterial LexA(BD)
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Functions of Activators Recruitment of General Transcription Factors Recruitment of RNA polymerase Two models of recruitment of pre-initiation complex; stepwise vs. holoenzyme
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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
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Recruitment of holoenzyme
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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
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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
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Action at a distance
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Looping effect can be mimicked by catenane formation
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Catenane experiments Catenated link between enhancer and test promoter (Ψ40) allowed activation
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Multiple enhancers Glucocorticoid RE basal level enhancer metal RE human metallothionine gene Modular arrangement of enhancers at sea urchin Endo16 gene
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Interaction between enhancers Combination of enhancer modules linked to CAT reporter tested in sea urchin development Organized regulations of six modules of many elements during development Different responses of enhancers in different environments
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Architectural Transcription Factors hTCRα 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 Looping is difficult – too close to the promoter
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Model of Enhanceosome
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Insulators DNA elements to block activation or repression from nearby elements GAGA boxes & binding protein, Trl in Drosophila, Enhancer blocking Barrier
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Mechanism of insulator activity ; Two hypothesis Looping model Sliding model
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Multiple insulator action
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Effect of insulators on gene expression
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Transcriptional Regulators Activators (repressors) 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
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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 Regulation of transcriptional factors
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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
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Signal transduction by PKA
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Co-activator ; CBP/p300
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Steroid receptor coactivator (SRC)
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Multiple roles of CBP/P300
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Protein degradation
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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
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7 major classes of cell surface receptors
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Signal transduction pathway involving Ras/Raf
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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)
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Activation of phospholipase C by GPCR Phosphatidiylinositol (PI) ; phosphorylated by PI kinase Phosphoinositide (PIP, PIP 2 ) ; cleaved by PLC to DAG and IP 3 IP 3 triggers release of Ca 2+ from ER DAG activates protein kinase C (PKC) Ca 2+ /calmodulin complex ; transcriptional regulation
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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
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Ca 2+ Short-lived increase/decrease in the concentration of certain low-molecular weight Intracelluar signaling molecules 2nd messenger
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Signal Transduction Cascade
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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
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