Structure and Function of Eukaryotic Transcription ActivatorsSlide 2Sp1: Factor for Upstream (Proximal) Class II Promoter ElementActivation DomainsDNA-binding domainsSlide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12bZIP proteinsSlide 14Slide 15Fig 12.13Function of Activation DomainsSlide 18GAL4 (which binds to an upstream element)Activation from a Distance: EnhancersFig. 12.20Slide 22Slide 23Combinatorial Transcription: expression and regulation depends on the combination of elements in the promoterInsulatorsRegulation of Transcription factors or “Regulating the Regulators”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 domainRegulation of galregulon in yeastRegulation ofamino acidbiosynthesis inyeastglucocorticoidreceptor: bindshormone and thenbinds DNA to altergene expressiongeneral upstreamactivator of pol IIgenes binds the GCbox.From Molecular Cell Biology 3rd edition, Lodish et al S cientific American Books 1995NCGal4GCN4GRSP1NNNCCCDNA binding domainActivation domainRecent 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 Domains1. 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 domains1. 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-sheet2 anti-parallel -sheetsturnalpha helix3 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 grooveDimerization domain - Coiled coil (helices)Fig. 12.4GAL4-DNA ComplexFig. 12.6Fig 12.6Fig. 12.7Glucocorticoid Receptor – DNA InteractionsWild-typeantennapedia- 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 12.9bZIP 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 leucinesFig. 12.10A Leucine Zipper is a Coiled Coil MotifPeptide from GCN4Fig. 12.11Fig 12.13Domain Independence demonstrated with a chimeric transcription factorFunction 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 modelsFactor binding to the enhancer induces:1. supercoiling2. sliding3. Looping4. TrackingFig. 12.20Models for enhancer functionTranscription 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 promoterFrom Fig. 12.22Looping out by a prokaryotic, enhancer-binding protein visualized by EM. NtrC – protein that binds glnA enhancer and RNAPσ54 polymerase – RNAP with a 54-kDa sigma factor (defective, needs enhancer)Like Fig. 9.20Combinatorial Transcription:expression and regulation depends on the combination of elements in the promoterGC boxMRE- metal response elementBLE- enhancer that responds to activator AP1GRE- Glucocorticoid response elementhuman metallothionine promoterFig. 12.23Insulators1. Block enhancers2. Also act as barriers to heterochromatin spreading induced by a silencerFig 12.28Regulation 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 destruction4. Sumoylation: covalent attachment of SUMO (small ubiquitin-like modifier) peptide to lysines, factor is inactivated but not destroyed5. Acetylation: histone acetyltransferases (HATs) acetylate lysines on histone and non-histone proteins, can be + or