Transcript 3. Activator, gene-specific transcription facotr

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