Transcription Regulation And Gene Expression in Eukaryotes UPSTREAM TRANSCRIPTION FACTORS
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1 Transcription Regulation And Gene Expression in Eukaryotes UPSTREAM TRANSCRIPTION FACTORS RG. Clerc March
2 UPSTREAM TRANSCRIPTION FACTORS Experimental approaches DNA binding domains (DBD) Transcription regulation domains Ligand binding domains
3 Typical gene and components involved in gene activation and inactivation Assembly of the RNA Pol II preinitiation complex and the major players in transcription regulation DBD is responsible for localizing a TA domain in the proximity of the basal apparatus
4 Modular structure of upstream transcription factors Transcription regulation Structural domain DBD DNA binding site (RE) GENE NH2 Structural domain DBD Transcription regulation COOH NH2 Structural domain DBD T regulation COOH
5 Modular structure of upstream transcription factors NH2 NH2 Structural domain X DBD Transcription regulation Structural domain DBD T regulation COOH X COOH domain swapping NH2 Structural domain DBD Transcription regulation COOH
6 Electrophoresis mobility shift assay (EMSA): a corner stone in deciphering DNA binding specificities
7 EMSA competition experiment DNA methylation interference assay
8 DNase I footprinting assay
9
10 Affinity chromatography to purify DNA:protein and protein:protein interactions
11 Southwestern: Expression cloning of DNA binding protein
12 TRX Factor Binding Assay on Random DNA oligomers: PCR Binding Site Selection
13 DNA Binding Domains DNA binding domain: the motif mindset concept. Recent invention as no one had anticipated that basic stuctural scaffolds would be used over and over again to recognize DNA and other proteins. Surprisingly large variety of protein familiy motifs involved in DNA sequence - specific recognition (promoter/enhancer contain short consensus sequences (RE) The role of DNA binding domains is to bring the transcription activation domain into the vincinity of the PIC
14 DNA Binding Domains (DBD) When a new protein was discovered, its gene cloned, the first questions were and to some extends still is: does it has any motifs we can understand? A helix-turn-helix? A zinc finger? An HMG domain? A β sheet DNA Motif? Uptream trx factors are grouped with respect to their respective highly conserved DNA binding domains In the case of DNA binding transcription factors the initial concept came from major stuctural determinations of bacterial regulators (lambda phage repressor, lambda Cro, Trp, 434 repressor, and CAP (catabolite activator protein from E. coli)
15 Helix-Turn-Helix Motif (HTH) Domain: high degree of homology between bacterial and eukaryotes DBD
16 Typically relatively short motifs are responsible for specific binding to DNA
17 Homeodomain-DNA Complex (PDB 1hdd) 5 Gln50 T Computer drawing of the tridimensional structure as determined by X-Ray crystallography of Engrailed homeodomain Ile47 Asn51 A A Arg3 T Arg5 T 3
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19 Homeodomain protein classes implicated in the mouse brain development
20 Body plan is constructed through interaction of the developmentally regulated homeotic genes posterior late low RA response anterior early high RA response trunk hindbrain
21 The POU domain: a large conserved region in the mammalian pit-1, oct1,2 and C. elegans unc96 gene products
22 POU domain DNA interaction 5 ATGCAAAT 3 (Octamer)
23 Can upstream transcription factors function as cell-cell signaling molecules? Homeodomain translocates across biological membranes
24 A zinc finger domain is a DBD Originally identified in TFIIIA (5S rrna), Zinc fingers form alpha helices that insert into the major groove associated with stabilizing beta sheets
25 A distinct form of Zn finger motifs in Nuclear Receptors
26 Glucocorticoid Receptor-DNA Complex (PDB 1glu) Zn finger K461 V462 R466 5 A G A A C A NNN T G T T C T3 T C T T G T NNN A C AA G A
27 Generation of DNA binding specificity mutants
28 bzip domain ( leucine zipper Leu spaced by 7 AA residues)
29 c-fos-c-jun-dna Complex (PDB 1fos) 5 T G A G T C A 3
30 Helix-Loop-Helix Domains: prototypical dimerizers Each amphipathic helix presents a face of hydrophobic residues on one side and charged residues on the other side
31 Polyamide motif for minor groove DNA sequence-specific recognition
32 DNA bending analysis by EMSA
33 LEF1 HMG Domain-DNA Complex (PDB 2lef) HELIX 3 HELIX 2 HELIX 1 Y76 L6 M10 M13 BASIC C-TERM E28 5 C C T T T G A A G C 3
34 LEF1 HMG Domain-DNA Complex (PDB 2lef) 5 C C T T T G A A G C 3
35 TBP : TATAA Binding protein
36 Number of Swissprot (SW) entries for the respective DNA binding folds A proteome-wide classification of DNAbinding protein families
37 Prototypical ligand binding domain of the nuclear hormone receptors mouse trap unliganded co-repressor binding liganded co-activator binding
38 Transactivation Domains VP16, GAL4 Acidic domains (DDD, EEE) SP1 Glutamine-rich domain (QQQXXXQQQ) CTF s Proline-rich domains (PPPXXXPPP) NTF s Isoleucine-rich domain IIXXII) Several motifs involved in transactivation in contrary to DBD, few structural data as largely unfolded motifs
39 Ser/Thr and Gln Transcription activation domains
40 HPV transactivation domain
41 Transrepression Domains Canonical transrepression domains in upstream transcription factors eg. Kruppel (KRAB box) are associated with strong transcription repression in eg. Kox-1 Targets co-repressor TIF1, widely distributed in transcriptional silencing NH2 Krab A Krab B 11 Zinc fingers Transcriptional repression of eukaryotic promoters by direct, or indirect (competitive binding), squelching, chromatin histone modifications etc COOH
42
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