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 TAF II 110 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.

3 views of C 2 H 2 Zinc fingers Often found as repeats in a protein. Bind in the major groove of DNA.

DNA-binding domain 1.2 Zn +2 bound by 6 cysteines 2.A Short  helix that docks into major groove Dimerization domain - Coiled coil (  helices) Fig GAL4- DNA Complex

Fig Fig 12.6

Fig 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 A Leucine Zipper is a Coiled Coil Motif Peptide from GCN4

Fig

Fig 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.supercoiling 2.sliding 3.Looping 4.Tracking

Fig Models 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- enhancer Psi40- rRNA promoter From Fig

Looping out by a prokaryotic, enhancer-binding protein visualized by EM. NtrC – protein that binds glnA enhancer and RNAP σ 54 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 box MRE- metal response element BLE- enhancer that responds to activator AP1 GRE- Glucocorticoid response element human metallothionine promoter Fig

Insulators 1.Block enhancers 2.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 -