Transcript Interaction
general: Activators protein-DNA interaction MBV4230 The sequence specific activators: transcription factors Modular design with a minimum of two functional domains 1. DBD - DNA-binding domain 2. TAD - transactivation domain DBD: several structural motifs classification into TF-families TAD - a few different types N Three classical categories Acidic domains (Gal4p, steroid receptor) Glutamine-rich domains (Sp1) Proline- rich domains (CTF/NF1) Mutational analyses - bulky hydrophobic more important than acidic Unstructured in free state - 3D in contact with target? DBD TAD Most TFs more complex Regulatory domains, ligand binding domains etc C MBV4230 TF classification based on structure of DBD Zinc finger bHelix-Loop-HelixTwo levels of recognition (Max) 1. Shape recognition Anhelix fits into the major groove in BDNA. This is used in most interactions 2. Chemical recognition Negatively charged sugar-phosphate chain involved in electrostatic interactions Hydrogen-bonding is crucial for sequence recognition Leucine zipper (Gcn4p) p53 DBD NFkB STAT dimer MBV4230 Alternative classification of TFs on the basis of their regulatory role Classification questions Is the factor constitutive active or requires a signal for activation? Does the factor, once synthesized, automatically enter the nucleus to act in transcription? If the factor requires a signal to become active in transcriptional regulation, what is the nature of that signal? Classification system I. Constitutive active nuclear factors II. Regulatory transcription factors Developmental TFs Signal dependent Steroid receptors Internal signals Cell surface receptor controlled Nuclear Cytoplasmic MBV4230 Classification - regulatory function Brivanlou and Darnell (2002) Science 295, 813 - MBV4230 Sequence specific DNA-binding - essential for activators TFs create nucleation sites in promoters for activation complexes Sequence specific DNA-binding crucial role Principles of sequence specific DNA-binding MBV4230 How is a sequence (cis-element) recognized from the outside? Shape recognition Chemical recognition Electrostatic interaction Form/ geometry Hydrogenbonds Hydrophobic interaction MBV4230 Complementary forms The dimension of anhelix fits the dimensions of the major groove in B-DNA Sidechains point outwards and are ideally positioned to engage in hydrogen bonds MBV4230 Direct reading of DNA-sequence Recognition of form The dimension of an -helix fits the dimensions of the major groove in B-DNA Most common type of interaction Usually multiple domains participate in recognition dimers of same motif tandem repeated motif Interaction of two different motifs recognition: detailed fit of complementary surfaces Hydration /vann participates seq specvariation of DNA-structure MBV4230 Example Steroid receptor MBV4230 Recognition by complementary forms 434 fag repressor MBV4230 DNAs form: B-DNA most common B-form Major groove Minor groove wide geometry fits -helix Each basepair with unique H-bondingpattern Deep and narrow geometry Each basepair binary H-bondingpattern B MBV4230 DNAs form: A-form more used in RNA-binding A-form Major groove Minor groove Deep and narrow geometry Wide and shallow A MBV4230 How is a sequence (cis-element) recognized from the outside? Shape recognition Chemical recognition Electrostatic interaction Form/ geometry Hydrogenbonds Hydrophobic interaction MBV4230 Next level: chemical recognition - reading of sequence information Negatively charged sugar-phosphate chain = basis for electrostatic interaction Equal everywhere - no sequencerecognition Still a main contributer to the strength of binding MBV4230 Electrostatic interaction Entropy-driven binding Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ - Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ - - Na+ Na+ Na+ Na+ - Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ - - Na+ Na+ Na+ Na+ Na+ Negative phosphate chain partially neutralized by a cloud of counter ions Na+ Na+ Na+ Na+ Na+ Counter ions liberated Entropy-driven binding MBV4230 How is a sequence (cis-element) recognized from the outside? Shape recognition Chemical recognition Electrostatic interaction Form/ geometry Hydrogenbonds Hydrophobic interaction MBV4230 Recognition by Hydrogen bonding Hydrogen-bonding is a key element in sequence specific recognition 10-20 x in contact surface Base pairing not exhausted in duplex DNA, free positions point outwards in the major groove D A A MBV4230 Unexploited H-bonding possibilities in the grooves Major groove AT-base pair Minor groove Major groove GC-base pair Minor groove MBV4230 A ”bar code” in the grooves Unique ”bar code” in major groove A D A AT-basepair A AT-basepair A Unique recognition of a base pair requires TWO hydrogen bonds In the major groove Binary ”bar code” in minor groove A D GC-basepair A GC-basepair A D AT-pair [AD-A] ≠ TA-pair [A-DA] GC-pair [AA-D] ≠ CG-pair [D-AA] A AT-pair [A-A] = TA-pair [A-A] GC-pair [ADA] = CG-pair [ADA] MBV4230 Docked prot side chains exploit the H-bonding possibilities for interaction Hydrogen-bonding is essential for sequence specific recognition 10-20 x in contact interphase Most contacts in major groove Purines most important A Zif example MBV4230 Interaction: Protein side chain - DNA bp Close up Amino acid sidechains points outwards from the -helix and are optimally positioned for base-interaction Still no ”genetic code” in the form of sidechain-base rules docking of the entire protein MBV4230 Interaction: Protein side chain - DNA bp Close up acid sidechains points outwards from the -helix and are optimally positioned for base-interaction Amino MBV4230 A network of H-bonds Example: c-Myb - DNA Protein DNA MBV4230 How is a sequence (cis-element) recognized from the outside? Shape recognition Chemical recognition Electrostatic interaction Form/ geometry Hydrogenbonds Hydrophobic interaction MBV4230 Hydrophobic contact points Ile Homeodomains MBV4230 The Homeodomain-family: common DBD-structure Homeotic genes - biology Regulation of Drosophila development Striking phenotypes of mutants - bodyparts move Control genetic developmental program Homeobox / homeodomain Conservered DNA-sequence “homeobox” in a large number of genes Encode a 60 aa “homeodomain” A stably folded structure that binds DNA Similarity with prokaryotic helix-turn-helix 3D-structure determined for several HDs Drosophila Antennapedia HD (NMR) Drosophila Engrailed HD-DNA kompleks (crystal) Yeast MAT2 MBV4230 Homeodomain-family: common DBD-structure Major groove contact via a 3 -helix structure helix 3 enters major groove (“recognition helix”) helix 1+2 antiparallel across helix 3 16 -helical aa conserved 9 in hydrophobic core some in DNA-contact interphase (common docking mechanism?) Positions important for sequence recognition N51 invariant: H-binding Adenine, role in positioning I47 (en, Antp) hydrophobic base contact Q50 (en), S50 (2) H-bond to Adenine, determining specificity R53 (en), R54 (2): DNA-contact MBV4230 Engrailed MBV4230 Antennapedia MBV4230 Homeodomain-family: common DBD-structure Minor groove contacted via N-terminal flexible arm R3 and R5 in engrailed and R7 in MAT2 contact AT in minor groove R5 conserved in 97% of HDs Deletions and mutants impair DNA-binding ftz HD (∆6aa N-term) 130-fold weaker DNA-binding MAT2 (R7A) impaired repressor POU (∆4,5) DNA-binding lost Loop between helix 1 and 2 determines Ubx versus Antp function Close to DNA exposed for protein protein interaction MBV4230 HD-paradox: what determines sequence specificity? Drosophila Ultrabithorax (Ubx), Antennapedia (Antp), Deformed (Dfd) and Sex combs reduced (Scr): closely similar HD, biological rolle very different Minor differences in DNA-binding in vitro TAAT-motif bound by most HD-factors contrast between promiscuity in vitro and specific effects in vivo Swaps reveal that surprisingly much of the specificity is determined by the N-terminal arm which contacts the minor groove Swaps: Antp with Scr-type N-term arm shows Scr-type specificity in vivo Swaps: Dfd with Ubx-type N-term arm shows Ubx-type specificity in vivo N-terminal arm more divergent than the rest of HD R5 and R7 (contacting DNA) are present in both Ubx, Antp, Dfd, and Scr Other tail aa diverge much more MBV4230 Solutions of the paradox Conformational effects mediated by N-term arm Even if the -helical HDs are very similar, a much larger diversity is found in the N-terminal arms that contact the minor groove Protein-protein interaction with other TFs through the N-terminal arm - enhanced affinity/specificity - the basis of combinatorial control MAT2 interaction with MCM1 - cooperative interactions Ultrabithorax- Extradenticle in Drosophila Hox-Pbx1 in mammals MBV4230 Combinatorial TFs give enhanced specificity TFs encoded by the the homeotic (Hox) genes govern the choice between alternative developmental pathways along the anterior– posterior axis. Hox proteins, such as Drosophila Ultrabithorax, have low DNA-binding specificity by themselves but gain affinity and specificity when they bind together with the homeoprotein Extradenticle (or Pbx1 in mammals). MBV4230 N-tail in protein-protein interaction - adopt different conformations HD b HD Mat-2/Mcm-1 Conformation determined by prot prot interaction MBV4230 The partner may also be a linker histone Repression of the mouse MyoD gene by the linker histone H1b and the homeodomain protein Msx1. The first evidence that a linker histone subtype operates in a genespecific fashion to regulate tissue differentiation MBV4230 It works impressively well Hox genes POU family MBV4230 POU-family: common DBD-structure The POU-name : A bipartite160 aa homeodomain-related DBD Pit-1 pituitary specific TF Oct-1 and Oct-2 lymphoide TFs Unc86 TF that regulates neuronal development in C.elegans a POU-type HD subdomain (C-terminally located) et POU-specific subdomain (N-terminally located) Coupled by a variabel linker (15-30 aa) POU is a structurally bipartite motif that arose by the fusion of genes encoding two different types of DNA-binding domain. MBV4230 POU: Two independent subdomains POUHD subdomain POUspec subdomain 60 aa closely similar to the classical HD Only weakly DNA-binding by itself (<HD) contacts 3´-half site (Oct-1: ATGCAAAT) docking similar to engrailed. Antp etc Main contribution to non-specific backbone contacts 75 aa POU-specific domain enhances DNA-affinity 1000x contacts 5´-half site (Oct-1: ATGCAAAT) contacts opposite side of DNA relative to HD structure similar to prokaryotic - and 434-repressors The two-part DNA-binding domain partially encircles the DNA. MBV4230 Flexible DNA-recognition POU-domains have intrinsic conformational flexibility and this feature appears to confer functional diversity in DNArecognition The subdomains are able to assume a variety of conformations, dependent on the DNA element. MBV4230 A POU prototype: Oct-1 Ubiquitously expressed Oct-1 (≠ cell type specific Oct-2) Oct-1 performs many divergent roles in cellular trx regulation partly owing to its flexibility in DNA binding and ability to associate with multiple and varied co-regulators Oct-1 activates transcription of genes that are involved in basic cellular processes Oct-1 activates small nuclear RNA (snRNA) and S-phase histone H2B gene transcription cell-specific promoters, particularly in the immune and nervous systems immunoglobulin (Ig) heavy- and lightchains Activate target genes by bidning to the “octamer” cis-element ATGCAAAT Hence the name “Octamer-motif binding protein” MBV4230 Flexibility On the natural highaffinity Oct-1 octamer (ATGCAAAT) binding site, the two Oct-1 POUsubdomains lie on opposite sides of the DNA The unstructured linker permits flexible subdomain positioning and hence diversity in Oct-1 sequence recognition. MBV4230 Oct-1: associates with multiple and varied co-regulators Oct-1 associates with a B-cell specific coregulator OCA-B (OBF-1). OCA-B stabilizes Oct-1 on DNA and provides a transcriptional activation domain. B-cell specific activation of immunoglobulin genes - for long a paradox Depended on octamer cis-elements B-cell express both ubiquitous Oct-1 and the cell type specific Oct-2 Hypothesis: Oct-2 aktivates IgGs (Wrong!) oct-2 deficient mouse normal development of early B-cells and cell lines without Oct-2 produce abundant amounts of Ig A B-cell specific coactivator mediates Oct-1 transactivation VP16 - a virus strategy to exploit a host TF MBV4230 Many viruses use Oct-1 to promote infection When herpes simplex virus (HSV) infects human cells, a virion protein called VP16, forms a trx regulatory complex with Oct-1 and the cell-proliferation factor HCF-1 VP16 = a strong transactivator, not itself DNA-binding, but becomes associated with DNA through Oct-1 The specificity of Oct-1 is altered from Octamer-seq to the virus cis-element TAATGARAT The VP16-induced complex has served as a model for combinatorial mechanisms of trx regulation Pax family MBV4230 Pax family Paired domain MBV4230 Paired domain DBD RED Major groove interaction: Minor groove interaction: Major groove interaction: Flex? PAI