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Transcription Factors…(TF) Transcription in eukaryote -controlled by trans-acting protein… TF -more complex than in prokaryotes Transcription Factors…(TF) TF Binding at cis-acting regulatory sequences Can be repressosr ar activators Located much farther from promoter 10-100 kb up/down stream Alternative controlled Alternative controlled Transthyretin (TTR)..steroid hormone transporter 2 control elements 1.85-2.okb upstream element 200 bp upstream Turn the gene on in two different cell types hepatocytes choroid plexus cells How to get the gene of TF 1. Detection of the controlled elements…5’ deletion assay 2. Biochemical isolation of TF -several chromatographic steps with cell extract …....get cognate proteins -Dnase footprint or mobility shift assay …....get specific DNA binding proteins -sequence-specific DNA affinity chromatography …...get a purified protein 3. In vivo transcription activation…to confirm protein function 4. Partial amino acid sequence determination and gene cloning 5. In vivo assay of the TF gene activity Detection of the controlled elements 5’-deletion analysis +++ +++ + + - Several chromatographic steps with cell extract …get cognate proteins Retardation assay Dnase footprint fractions collected DNase footprint/mobility shift assay Biochemical isolation of TF DNase footprint assay Interesting fractions = 9-12 Biochemical isolation of TF Mobility shift assay Gel retardation assay Sequence-specific DNA affinity chromatography …...get a purified protein fractions 9-12 Specific regulatory sequence In vivo transcription activation assay purified protein In vitro transcription activation…to confirm protein function SP1 binding site in SV40 genome No SP1 binding site on adenovirus SP1 protein has no effect on transcription from the adenovirus promoterbut stimulate transcription from SV40 promoter about 10 folds. SP1 RNA -+ -+ isolated TF partial protein sequences oligonucleotide probe TF gene To test the gene function….in vivo assay General structure of TF Study by in vitro translation and N-terminal deletion assay 2 functional domains found UAS = upstream activating sequence General Structure of TF 2 functional domains DNA –binding domain…interact with specific DNA sequence Activation domain…interact with other proteins 5 structural types of DNA binding domain 1. Homeodomain (helix-turn-helix) 2. Zinc-finger protein 3. Winged-helix (fork-head) 4. Leucine zipper 5. Helix-loop-helix Homeodomain ( helix-turn-helix ) The name derived from a group of Drosophila genes in which the conserved sequence encoding this structural domain was first noted. Homeotic genes, mutation in these genes resulted in the transformation of one body part into another during the development. Two of most-studied of these genes Antennapedia (Antp) Ultrabithorax (Ubx) normal fly mutant fly Structure of homeodomain Helix-Turn-Helix protein and Homeoboxes -Three -helical regions separated by short turns. -Bind to DNA in similar manner. -Bind as dimers -Amino acid substitution in the helices near C terminal disturb the binding in major groove of DNA -The region encoding about 60 amino acids was remarkably conserved in all these genes called homeobox. Homeobox = region in the gene Homeotic gene = gene Homeodomain / helix-turn-helix protein = protrein encoded by homeotic gene Example of helix-turn-helix proteins Gene product Organism Functions MAT 1 MAT 2 MAT a1 Antennapedia Ultrabithorax Engrailed Paired Fushi tarazu HOX Unc86 Oct1 Oct2 Pit activates -specific genes inactivates a-specific genes yeast yeast yeast Drosophila Drosophila Drosophila Drosophila Drosophila mouse C. elegans human human mouse combined with 2 repressed haploid-specific genes homeotic gene homeotic gene segment-polarity gene pair-rule gene pair-rule gene developmental regulators determine cell lineage in worms generally distributed activator lymphoid-specific activator pituitary-specific activator Determine the fate of each segments (mouth, legs, wings,etc.) The 9 homeotic genes in fly Bithorax complex (Bx-C) Ultrabithorax (Ubx) Abdominal A (abdA) Abdominal B (abdB) Antennapedia complex (Ant-C) labial (lab) Prodoscipedia (pb) Deformed (Dfd) Sex combs reduced (Scr) Antennapedia (Antp) Engrailed Example of homeotic genes in mouse and human Pit-1 mutant Growth hormone deficiency Example of homeotic genes in plant 3 classes of floral organ-identity genes in Arabidopsis, A B and C, encoded TF of MADS family