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 transporter2 control elements1.85-2.okb upstream element200 bp upstream Turn the gene on in two different cell types

hepatocyteschoroid plexus cells

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

Detection of the controlled

elements

5’-deletion analysis

+++ +++ + + -

fractions collected

DNase footprint/mobility shift assay

Several chromatographic steps with cell extract

…get cognate proteins

Retardation assay Dnase footprint

DNase footprint assay

Biochemical isolation of TF

Interesting fractions = 9-12

Mobility shift assay Gel retardation assay

Biochemical isolation of TF

fractions 9-12

purified protein

Sequence-specific DNA affinity chromatography

…...get a purified proteinSpecific regulatory sequence

In vivo transcription activation assay

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.

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 TF2 functional domainsDNA –binding domain…interact with specific DNA sequenceActivation 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 geneHomeotic gene = geneHomeodomain / helix-turn-helix protein = protrein encoded by homeotic gene

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

Determine the fate of each segments (mouth, legs, wings,etc.)

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

Example of homeotic genes in mouse and human

Pit-1 mutantGrowth 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