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