Ch 12. Transcription Activators in Eukaryotes Gene-specific transcription factor
Jan 18, 2016
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); 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 + Zntwo C in -ribbon, two H in -helix, basic a.a. in -helix
NMR study of Xfin finger
TFIIIA
9 repeats of 30-residues
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
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 grooveRecognition 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
hsp90Type I : glucocorticoid RcType II : thyroid Rc & RXR (in nucleus) activator vs. repressorType III : orphan receptor
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 FactorsRecruitment 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 transcriptionVP16-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 holoenzymerecruitment model
Interaction among activators: dimerization
Increase affinity, specificity, additional regulation
Jun, Fos: bZIP family
Dimerization; Jun+Fos= AP1TRE (TPA response element)
Jun homodimer - weak bindingJun-Fos - strong bindingFos dimer - no binding
Action at a distance
Looping effect can be mimicked by catenane formation
Catenane experiments
Catenated link between enhancer and testpromoter (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
Combination of enhancer modules linked to CAT reportertested in sea urchin development
Organized regulations of six modules of many elements during development Different responses of enhancers in different environments
Architectural Transcription Factors
hTCRα
LEF-1 (Lymphoid enhancer binding factor) no activation by itself, only help activation by other factorsHMG domain; binds to minor groove & bends DNA Transcription factors bind close together
Looping is difficult – too close to the promoter
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
Looping model
Sliding model
Multiple insulator action
Effect of insulators on gene expression
TranscriptionalRegulatorsActivators
(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
• 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
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
Ca2+Short-lived increase/decrease in the concentration of certain low-molecular weightIntracelluar signaling molecules
2nd messenger
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