Transcription Factors … (TF)
description
Transcript of Transcription Factors … (TF)
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Transcription Factors…(TF)Transcription in eukaryote-controlled by trans-acting protein… TF-more complex than in prokaryotes
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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
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Alternative controlled
Transthyretin (TTR)..steroid hormone transporter2 control elements1.85-2.okb upstream element200 bp upstream Turn the gene on in two different cell types
hepatocyteschoroid plexus cells
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How to get the gene of TF1. Detection of the controlled elements…5’ deletion assay2. 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 function4. Partial amino acid sequence determination and gene cloning5. In vivo assay of the TF gene activity
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Detection of the controlled
elements
5’-deletion analysis
+++ +++ + + -
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fractions collected
DNase footprint/mobility shift assay
Several chromatographic steps with cell extract
…get cognate proteins
Retardation assay Dnase footprint
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DNase footprint assay
Biochemical isolation of TF
Interesting fractions = 9-12
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Mobility shift assay Gel retardation assay
Biochemical isolation of TF
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fractions 9-12
purified protein
Sequence-specific DNA affinity chromatography
…...get a purified proteinSpecific regulatory sequence
In vivo transcription activation assay
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In vitro transcription activation…to confirm protein function
SP1 binding site in SV40 genome
No SP1 binding site on adenovirus
SP1 RNA - + - +
SP1 protein has no effect on transcription from the adenovirus promoterbut stimulate transcription from SV40 promoter about 10 folds.
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isolated TF
partial protein sequences
oligonucleotide probe
TF gene
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To test the gene function….in vivo assay
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General structure of TF
Study by in vitro translation and N-terminal deletion assay
2 functional domains found
UAS = upstream activating sequence
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General Structure of TF2 functional domainsDNA –binding domain…interact with specific DNA sequenceActivation domain…interact with other proteins
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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
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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
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Structure of homeodomain
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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 geneHomeotic gene = geneHomeodomain / helix-turn-helix protein = protrein encoded by homeotic gene
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Example of helix-turn-helix proteinsGene product Organism FunctionsMAT 1 yeast activates -specific genesMAT 2 yeast inactivates a-specific genesMAT a1 yeast combined with 2 repressed haploid-specific genesAntennapedia Drosophila homeotic geneUltrabithorax Drosophila homeotic geneEngrailed Drosophila segment-polarity genePaired Drosophila pair-rule geneFushi tarazu Drosophila pair-rule geneHOX mouse developmental regulatorsUnc86 C. elegans determine cell lineage in wormsOct1 human generally distributed activatorOct2 human lymphoid-specific activatorPit mouse pituitary-specific activator
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Determine the fate of each segments (mouth, legs, wings,etc.)
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The 9 homeotic genes in flyBithorax 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
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Example of homeotic genes in mouse and human
Pit-1 mutantGrowth hormone deficiency
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Example of homeotic genes in plant
3 classes of floral organ-identity genes in Arabidopsis, A B and C, encoded TF of MADS family