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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