Structure and Function of Eukaryotic
Transcription Activators
•
Many have modular structure:
1. DNA-binding domain
2. Transcription activating domain
•
•
Proteins can have > 1 of each, and
they can be in different positions in
protein.
Many also have a dimerization domain
Recent data suggests SP1 actually has 4 activating domains.
Sp1: Factor for Upstream (Proximal)
Class II Promoter Element
• Binds GC boxes,
stimulates transcription
• Interacts with TAFII110
in TFIID
• Also stimulates
transcription of TATAless class II promoters
(by promoting TFIID
binding)
Activation Domains
1. Acidic (e.g., GAL4, 49 aa domain – 11
acidic aa)
2. Glutamine-rich (e.g., 2 in Sp1, ~25%
gln)
3. Proline-rich (e.g., CTF, 84 aa domain
– 19 are proline)
DNA-binding domains
1. Zinc–containing motifs
– Zinc fingers (Sp1 and TFIIIA)
– Zinc modules (GR and other nuclear
receptors)
– Modules with 2 Zinc ions and 6
cysteines (GAL4)
2. Homeodomains - 60-aa domains originally
found in homeotic mutants
3. bZIP and bHLH motifs - a highly basic
DNA-binding domain and a dimerization
domain (leucine zipper or helix-loop-helix)
Amino acid
side chains
in proteins
can form Hbonds to
DNA bases.
Critical for
sequencespecific
binding to
DNA.
3 views of C2H2 Zinc fingers
Often found as
repeats in a protein.
Bind in the major
groove of DNA.
GAL4DNA
Complex
Dimerization
domain Coiled coil (a
helices)
Fig. 12.4
DNA-binding domain
1. 2 Zn+2 bound by
6 cysteines
2. A Short a helix
that docks into
major groove
Fig. 12.6
Fig 12.6
Fig. 12.7
Glucocorticoid Receptor – DNA Interactions
- Homeotic mutants have wrong organs (organ-identity
mutants)
- Occur in animals and plants
- Important regulatory genes
Wild-type
“Here’s looking at you”
antennapedia
12.9
• Homeotic genes are
transcription factors!
• Have a conserved
DNA-binding domain
(Homeodomain) that
resembles a helixloop-helix (HLH)
domain.
• Bind as a monomer
bZIP proteins
• Have DNA binding and
dimerization domains
• DNA binding region is
very basic (R and K
residues)
• Dimerization involves a
Leucine Zipper
• Can form heterodimers!
Alpha helices form a coiled-coil with interdigitating leucines
A Leucine
Zipper is a
Coiled Coil
Motif
Peptide from
GCN4
Fig. 12.10
Fig. 12.11
Domain Independence demonstrated with a
chimeric transcription factor
Fig 12.13
Function of Activation Domains
• Recruit specific components of the pre-initiation
complex (a), or the holoenzyme (b).
Holoenzyme or Component Recruitment?
GAL4
(which binds to an upstream element)
1. Promotes binding of TFIIB, which promotes
recruitment of the other factors and RNAP.
–
Probably binds directly to TFIIB (i.e., it doesn’t
work by stimulating TFIID to bind TFIIB tighter)
2. GAL4 also promotes assembly of
downstream basal factors, TFIIE and/or
TFIIF+RNAP II.
Activation from a Distance:
Enhancers
•
There are at least 4 possible models
Factor binding to the enhancer induces:
1. supercoiling
2. sliding
3. Looping
4. Tracking
Models for enhancer function
Fig. 12.20
E- enhancer
Psi40- rRNA promoter
Transcription of DNAs 1-5 was tested in Xenopus oocytes.
Results: good transcription from 2, 3, and 4 (also 2 >3 or 4) but not 5.
Conclusion: Enhancer does not have to be on same DNA molecule, but
must be somewhat close.
Rules out the sliding and supercoiling models.
From Fig. 12.22
Looping out by a
prokaryotic,
enhancer-binding
protein visualized by
EM.
NtrC – protein that
binds glnA enhancer
and RNAP
σ54 polymerase –
RNAP with a 54-kDa
sigma factor (defective,
needs enhancer)
Like Fig. 9.20
Combinatorial Transcription:
expression and regulation depends on the
combination of elements in the promoter
human metallothionine promoter
GC box
MRE- metal response element
BLE- enhancer that responds to activator AP1
GRE- Glucocorticoid response element
Fig. 12.23
Insulators
1.
2.
Block enhancers
Also act as barriers to heterochromatin spreading
induced by a silencer
Fig 12.28
Regulation of Transcription factors
or “Regulating the Regulators”
A lot of post-translational regulation: Why?
- Quicker response time
- Avoid silencing by keeping the transcription factor gene on (?)
Some of the mechanisms:
1. Coactivators or mediators
2. Phosphorylation-dephosphorylation: can be + or 3. Ubiquitination (deubiquitination): covalent attachment of ubiquitin
(small protein) to lysines can modulate activity or trigger destruction
4. Sumoylation: covalent attachment of SUMO (small ubiquitin-like
modifier) peptide to lysines, factor is inactivated but not destroyed
5. Acetylation: histone acetyltransferases (HATs) acetylate lysines
on histone and non-histone proteins, can be + or -