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

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.

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)

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 H-bonds to DNA bases.

Critical for sequence-specific binding to DNA.

.

-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.

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

Fig. 12.6

Fig 12.6

Fig. 12.7

Glucocorticoid Receptor – DNA Interactions

Wild-type antennapedia

- Homeotic mutants have wrong organs (organ-identity mutants)

- Occur in animals and plants- Important regulatory genes

“Here’s looking at you”

• 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

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

Fig. 12.10

A Leucine Zipper is a Coiled Coil Motif

Peptide from GCN4

Fig. 12.11

Fig 12.13

Domain Independence demonstrated with a chimeric transcription factor

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. supercoiling2. sliding3. Looping4. Tracking

Fig. 12.20Models for enhancer function

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

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

GC boxMRE- metal response elementBLE- enhancer that responds to activator AP1GRE- Glucocorticoid response element

human metallothionine promoter

Fig. 12.23

Insulators1. Block enhancers2. Also act as barriers to heterochromatin spreading

induced by a silencer

Fig 12.28

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 -