Molecular Biology

Chapter 12 Outline

Domains within transcription activators:

∑ Transcription activators must have an activation domain and a DNA binding domain; some activators also have dimerization and ligand binding domains.

∑ DNA binding domains include three types of zinc-containing modules, homeodomains and bZIP/bHLH motifs.

∑ Transcription activation domains include acidic domains, glutamine rich domains and proline rich domains.

DNA binding domain structures:

∑ Zinc containing DNA binding domains

a) Zinc fingers

Comprised of an alpha helix and a beta strand; Zn binds two histidines in the alpha helix and two cysteines in the beta strand (F12.1); Basic amino acids in the alpha helix contact nucleotides in the major groove (F12.3); Each of the three fingers from Zif268 contact nucleotides and phosphates via basic residues (F12.4); The beta strand of Zn fingers positions the alpha helix in the major groove; Zn finger proteins with multiple fingers typically bind as monomers.

b) Gal4

Binds a upstream activating sequences (UASs) as a dimer; uses six cysteines and two Zn ions to bind DNA; Basic residues in the alpha helix makes contacts with bases in the major groove (F12.5, F12.6); Dimerization motif comprised of a coiled coil alpha helix.

c) Nuclear receptors

Interact with hormone ligands and bind to hormone response elements to activate transcription; One class is in normally bound to hsp90 in the cytoplasm until hormone is bound, then hsp90 is released and the receptors moves to the nucleus to bind its target sequence as a dimer (F12.7); Another class is always in the nucleus and binds target sequence and represses transcription in the absence of hormone, but activates transcription in the presence of hormone; Binding occurs at half sites separated by 3bp using the recognition alpha helix (F12.8).

∑ Homeodomains

Comprised of three alpha helices where the first two form a helix-turn-helix and the 3rd is the recognition helix (F12.11); They have weak binding specificity on their own.

∑ bZIP and bHLH domains

Both domains combine DNA binding and dimerization; the ≥b≤ stands for the basic DNA binding; The ZIP domain is an alpha helix having leucines every seven amino acids so they lie on the same side of the helix; Dimerization is mediated by a coiled coil formed by interactions among leucines (F12.12); bZIP binds DNA like forceps (F12.14); bHLH also forms dimers via alpha helix interactions which position the basic region in the major groove (F12.15).

Independence of domains

∑ Activation and DNA binding domains are modular and can be swapped to change the specificity of binding and activation or repression (F12.16).

Activator function

∑ Activators function to recruit preinitiation complex factors to the promoter; recruitment can occur stepwise or as holoenzymes (F12.17).

∑ Recruitment of TFIID via the VP16 activation domain; TFIID binds to the VP16 activation domain (F12.18).

∑ Recruitment of TFIIB by the Gal4 activation domain; Use of E4 promoter with a Gal4 site to bind various core transcription factors (TFs) either before or after Gal4 binding (F12.19); Gal4 preincubation with core TFs stimulates transcription, but if TFs are incubated without Gal4 and washed, there is no transcription (F12.20); TFIID does not require Gal4 bind, but TFIIB (and all subsequent core TFs) requires Gal4 (F12.21); TFIIB can not add without TFIID; Assay for addition of factors to the E4 + Gal4 promoter using antibodies (F12.22); Gal4 and TAFs are required to recruit PolII, TFIIF and TFIIE (F12.22); Multiple UAS enhancers cooperate to stimulate transcription through recruitment of TFIIE by Gal4 (F12.23); Other activation domains recruit TFIIB and TFIIE to the promoter.

∑ Core TFs can be recruited to the promoter as a holoenzyme by transcription activators (F12.24); Recruitment can occur through any part of the activator that can bind to the core TFs, not just the activation domain (F12.25).

Interaction among activators

∑ Dimerization of activators increases their affinity for binding sites four fold (i.e. the square of the number of contacts); Activators are typically in low quantities; Electrophoretic mobility shift assays (EMSAs) show that Fos-Jun heterodimers bind with higher affinity than homodimers (F12.26).

∑ Four models for how activators can act at a distance (F12.27); Experiments using catenanes show that enhancers do not have to be on the same piece of DNA (F12.28, F12.29).

∑ Multiple enhancers act in concert with activators at different developmental stages or in different cell types to control gene expression (F12.31, F12.32).

∑ Architectural TFs activate transcription by altering the shape of DNA so that TFs that bind enhancers can properly interact; LEF-1 is an HMG protein that can not activate by itself, but binds to DNA and alters its conformation so that Ets-1 and CREB can stimulate transcription (F12.33, F12.34); HMG1 can not activate IFNbeta alone, but binds along with other activators to form an enhanceosome (F12.35, F12.36); enhanceosome formation is cooperative.

∑ Insulators can block function of enhancer and silencers so they donπt act on inappropriate genes (F12.38); An insulator in Drosophila is encoded by the Trl gene which binds to GAGA sequences; The mechanism of insulator function is not known.

∑ Interference of one activator by another is called squelching; Squelching caused by competition for binding a scarce factor called a mediator; Mediators only work in conjunction with an activator; Mediator function with Gal4-VP16 (F12.40); CBP is a mediator (also called a co-activator) that binds only to phosphorylated or ligand bound activators; Activators subject to CBP mediated co-activation are CREB (via PKA phosphorylation) (F12.41), nuclear receptors bound to ligand (in conjunction with steroid receptor coactivators, or SRCs) (F12.42), and AP-1/Sap-1a (via MAPK phosphorylation)B (F12.43); In addition to activation by recruiting core TFs, CBP also opens up DNA by acetylating histones.

Regulation of transcription factors

∑ Transcription factor function can be regulated in five ways: (1) Nuclear receptors change from repressor to activator upon ligand binding, (2) Nuclear receptor binding to ligand causes dissociation of an inhibitor and movement into the nucleus, (3) Phosphorylation can lead to binding of mediators, (4) Phosphorylation of some inhibitors leads to their degradation and allows the released activators to activate transcription, and (5) Phosphorylation of activators leads to their degradation, thus inactivating the kinases leads transcriptional activation.

∑ Phosphorylation of CREB, Jun (an AP-1 component), IkB and beta-catenin result from signal transduction pathways; The MAPK signal induction pathway from growth factor binding to AP-1 induced gene activation (F12.44); Amplification of signaling.