structure and function of eukaryotic transcription activators
DESCRIPTION
Structure and Function of Eukaryotic Transcription Activators. Many have modular structure: DNA-binding domain Transcription activating domain Proteins can have > 1 of each, and they can be in different positions in protein. Many also have a dimerization domain. - PowerPoint PPT PresentationTRANSCRIPT
![Page 1: Structure and Function of Eukaryotic Transcription Activators](https://reader034.vdocuments.mx/reader034/viewer/2022051020/56815c34550346895dca188f/html5/thumbnails/1.jpg)
Structure and Function of Eukaryotic Transcription Activators
• Many have modular structure:1. DNA-binding domain2. Transcription activating domain
• Proteins can have > 1 of each, and they can be in different positions
in protein. • Many also have a dimerization domain
![Page 2: Structure and Function of Eukaryotic Transcription Activators](https://reader034.vdocuments.mx/reader034/viewer/2022051020/56815c34550346895dca188f/html5/thumbnails/2.jpg)
Regulation of galregulon in yeast
Regulation ofamino acidbiosynthesis inyeast
glucocorticoidreceptor: bindshormone and thenbinds DNA to altergene expression
general upstreamactivator of pol IIgenes binds the GCbox.
From Molecular Cell Biology 3rd edition, Lodish et al Scientific American Books 1995
N C
Gal4
GCN4
GR
SP1
N
N
N
C
C
C
DNA binding domainActivation domain
Recent data suggests SP1 actually has 4 activating domains.
![Page 3: Structure and Function of Eukaryotic Transcription Activators](https://reader034.vdocuments.mx/reader034/viewer/2022051020/56815c34550346895dca188f/html5/thumbnails/3.jpg)
Sp1: Factor for Upstream (Proximal) Class II Promoter Element
• Binds GC boxes, stimulates transcription
• Interacts with TAFII110 in TFIID
• Also stimulates transcription of TATA-less class II promoters (by promoting TFIID binding)
![Page 4: Structure and Function of Eukaryotic Transcription Activators](https://reader034.vdocuments.mx/reader034/viewer/2022051020/56815c34550346895dca188f/html5/thumbnails/4.jpg)
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)
![Page 5: Structure and Function of Eukaryotic Transcription Activators](https://reader034.vdocuments.mx/reader034/viewer/2022051020/56815c34550346895dca188f/html5/thumbnails/5.jpg)
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)
![Page 6: Structure and Function of Eukaryotic Transcription Activators](https://reader034.vdocuments.mx/reader034/viewer/2022051020/56815c34550346895dca188f/html5/thumbnails/6.jpg)
Amino acid side chains in proteins can form H-bonds to DNA bases.
Critical for sequence-specific binding to DNA.
![Page 7: Structure and Function of Eukaryotic Transcription Activators](https://reader034.vdocuments.mx/reader034/viewer/2022051020/56815c34550346895dca188f/html5/thumbnails/7.jpg)
.
-sheet
-sheet
2 an
ti-pa
ralle
l -sh
eets
turnalpha helix
3 views of C2H2 Zinc fingers
Often found as repeats in a protein.Bind in the major groove of DNA.
![Page 8: Structure and Function of Eukaryotic Transcription Activators](https://reader034.vdocuments.mx/reader034/viewer/2022051020/56815c34550346895dca188f/html5/thumbnails/8.jpg)
DNA-binding domain1. 2 Zn+2 bound by
6 cysteines2. A Short helix
that docks into major groove
Dimerization domain -
Coiled coil (helices)
Fig. 12.4
GAL4-DNA Complex
![Page 9: Structure and Function of Eukaryotic Transcription Activators](https://reader034.vdocuments.mx/reader034/viewer/2022051020/56815c34550346895dca188f/html5/thumbnails/9.jpg)
Fig. 12.6
Fig 12.6
![Page 10: Structure and Function of Eukaryotic Transcription Activators](https://reader034.vdocuments.mx/reader034/viewer/2022051020/56815c34550346895dca188f/html5/thumbnails/10.jpg)
Fig. 12.7
Glucocorticoid Receptor – DNA Interactions
![Page 11: Structure and Function of Eukaryotic Transcription Activators](https://reader034.vdocuments.mx/reader034/viewer/2022051020/56815c34550346895dca188f/html5/thumbnails/11.jpg)
Wild-type antennapedia
- Homeotic mutants have wrong organs (organ-identity mutants)
- Occur in animals and plants- Important regulatory genes
“Here’s looking at you”
![Page 12: Structure and Function of Eukaryotic Transcription Activators](https://reader034.vdocuments.mx/reader034/viewer/2022051020/56815c34550346895dca188f/html5/thumbnails/12.jpg)
• Homeotic genes are transcription factors!
• Have a conserved DNA-binding domain (Homeodomain) that resembles a helix-loop-helix (HLH) domain.
• Bind as a monomer
12.9
![Page 13: Structure and Function of Eukaryotic Transcription Activators](https://reader034.vdocuments.mx/reader034/viewer/2022051020/56815c34550346895dca188f/html5/thumbnails/13.jpg)
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 inter-digitating leucines
![Page 14: Structure and Function of Eukaryotic Transcription Activators](https://reader034.vdocuments.mx/reader034/viewer/2022051020/56815c34550346895dca188f/html5/thumbnails/14.jpg)
Fig. 12.10
A Leucine Zipper is a Coiled Coil Motif
Peptide from GCN4
![Page 15: Structure and Function of Eukaryotic Transcription Activators](https://reader034.vdocuments.mx/reader034/viewer/2022051020/56815c34550346895dca188f/html5/thumbnails/15.jpg)
Fig. 12.11
![Page 16: Structure and Function of Eukaryotic Transcription Activators](https://reader034.vdocuments.mx/reader034/viewer/2022051020/56815c34550346895dca188f/html5/thumbnails/16.jpg)
Fig 12.13
Domain Independence demonstrated with a chimeric transcription factor
![Page 17: Structure and Function of Eukaryotic Transcription Activators](https://reader034.vdocuments.mx/reader034/viewer/2022051020/56815c34550346895dca188f/html5/thumbnails/17.jpg)
Function of Activation Domains• Recruit specific components of the pre-initiation
complex (a), or the holoenzyme (b).
![Page 18: Structure and Function of Eukaryotic Transcription Activators](https://reader034.vdocuments.mx/reader034/viewer/2022051020/56815c34550346895dca188f/html5/thumbnails/18.jpg)
Holoenzyme or Component Recruitment?
![Page 19: Structure and Function of Eukaryotic Transcription Activators](https://reader034.vdocuments.mx/reader034/viewer/2022051020/56815c34550346895dca188f/html5/thumbnails/19.jpg)
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.
![Page 20: Structure and Function of Eukaryotic Transcription Activators](https://reader034.vdocuments.mx/reader034/viewer/2022051020/56815c34550346895dca188f/html5/thumbnails/20.jpg)
Activation from a Distance: Enhancers
• There are at least 4 possible models
Factor binding to the enhancer induces:1. supercoiling2. sliding3. Looping4. Tracking
![Page 21: Structure and Function of Eukaryotic Transcription Activators](https://reader034.vdocuments.mx/reader034/viewer/2022051020/56815c34550346895dca188f/html5/thumbnails/21.jpg)
Fig. 12.20Models for enhancer function
![Page 22: Structure and Function of Eukaryotic Transcription Activators](https://reader034.vdocuments.mx/reader034/viewer/2022051020/56815c34550346895dca188f/html5/thumbnails/22.jpg)
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.
E- enhancerPsi40- rRNA promoter
From Fig. 12.22
![Page 23: Structure and Function of Eukaryotic Transcription Activators](https://reader034.vdocuments.mx/reader034/viewer/2022051020/56815c34550346895dca188f/html5/thumbnails/23.jpg)
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
![Page 24: Structure and Function of Eukaryotic Transcription Activators](https://reader034.vdocuments.mx/reader034/viewer/2022051020/56815c34550346895dca188f/html5/thumbnails/24.jpg)
Combinatorial Transcription:expression and regulation depends on the combination of elements in the promoter
GC boxMRE- metal response elementBLE- enhancer that responds to activator AP1GRE- Glucocorticoid response element
human metallothionine promoter
Fig. 12.23
![Page 25: Structure and Function of Eukaryotic Transcription Activators](https://reader034.vdocuments.mx/reader034/viewer/2022051020/56815c34550346895dca188f/html5/thumbnails/25.jpg)
Insulators1. Block enhancers2. Also act as barriers to heterochromatin spreading
induced by a silencer
Fig 12.28
![Page 26: Structure and Function of Eukaryotic Transcription Activators](https://reader034.vdocuments.mx/reader034/viewer/2022051020/56815c34550346895dca188f/html5/thumbnails/26.jpg)
Regulation of Transcription factorsor “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 mediators2. 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 -