Transcription Factors and Gene Control
Characteristics of transcription factors
- Are modular proteins composed of at least two distinct functional
domains
- DNA-binding domain - recognises and binds to specific DNA
control elements, usually one per TF
- Regulatory domain - interacts with other proteins to
stimulate/repress transcription of a promoter, may be more than
one per TF
- Are +/- 100 nt
- Motif - are alpha-helices and beta sheets with loops that connect them
e.g. helix-loop-helix
- Domain - different motifs bind to each other and form a tertiary
structure which has a specific modular function e.g. DNA binding
Isolating a TF to a known DNA sequence
- Use DNA affinity chromatography
- Purify the TF by making use of the fact that it binds very specifically to
its DNA binding site
- Requirements:
- Nucleotide sequence of specific DNA-binding site of TF
- Need to obtain a small amount of purified TF
- The purified TF obtained can be used in further studies
- The protein extract is loaded into a column containing resin (e.g.
sepharose)
- A logan (DNA oligo) is attached to the resin
- The ligand binds to the target protein and nonspecific protein pass
through
,Process of DNA affinity chromatography
- Synthesise oligo-nt of DNA binding site and link to the sepharose
column
- Pass cellular or nuclear extract through the column which contains the
TFs
- The TF protein will bind to its DNA recognition site
- Release and elute the TF from the column using high-salt buffer wash
- Obtain partial AA sequence of TF protein by mass spectrometry
- Search a protein database for a match to the partial sequence
(bioinformatics approach)
DNA-binding domains
- TF are classified based on the type of structural motifs/domains they
use to bind to the control elements
- Helix-turn-helix motif
- Homeodomain
- Zinc fingers
- Leucine zipper domain
- Helix-loop-helix domain
- DNA-binding domains have characteristic consensus amino acid
sequences
- Many TFs bind in the form of homodimers (two identical protein
molecules) or heterodimers (two different molecules)
- Amino acids of TF domain enables it to very specifically recognise and
bind to nucleotides of a specific DNA element
, Helix-turn-helix motif
- Is one of the most common structural domains (e.g. homeotic/Hox TFs)
- Consists of two alpha-helices connected by a short chain of amino
acids that forms a beta sheet turn
- The two helices are held at a fixed angle
- One alpha helix is involved in nucleotide-specific recognition - called
the recognition helix
- This lies across the major groove of DNA and directly contacts
nucleotides
- Characteristics
- Other helix helps position the recognition helix correctly and
stabilises it
- TFs with this domain bind to DNA as homodimers
- Additional motifs (e.g. alpha helix) or regulatory domains may be
present on the protein
Homeotic TFs
- Have a homeodomain with helix-turn-helix motifs
- Homeotic genes encode homeotic TFs
- Homeobox is a coding nt sequence within the homeotic genes
- This encodes the homeodomain as part of the protein
Characteristics of transcription factors
- Are modular proteins composed of at least two distinct functional
domains
- DNA-binding domain - recognises and binds to specific DNA
control elements, usually one per TF
- Regulatory domain - interacts with other proteins to
stimulate/repress transcription of a promoter, may be more than
one per TF
- Are +/- 100 nt
- Motif - are alpha-helices and beta sheets with loops that connect them
e.g. helix-loop-helix
- Domain - different motifs bind to each other and form a tertiary
structure which has a specific modular function e.g. DNA binding
Isolating a TF to a known DNA sequence
- Use DNA affinity chromatography
- Purify the TF by making use of the fact that it binds very specifically to
its DNA binding site
- Requirements:
- Nucleotide sequence of specific DNA-binding site of TF
- Need to obtain a small amount of purified TF
- The purified TF obtained can be used in further studies
- The protein extract is loaded into a column containing resin (e.g.
sepharose)
- A logan (DNA oligo) is attached to the resin
- The ligand binds to the target protein and nonspecific protein pass
through
,Process of DNA affinity chromatography
- Synthesise oligo-nt of DNA binding site and link to the sepharose
column
- Pass cellular or nuclear extract through the column which contains the
TFs
- The TF protein will bind to its DNA recognition site
- Release and elute the TF from the column using high-salt buffer wash
- Obtain partial AA sequence of TF protein by mass spectrometry
- Search a protein database for a match to the partial sequence
(bioinformatics approach)
DNA-binding domains
- TF are classified based on the type of structural motifs/domains they
use to bind to the control elements
- Helix-turn-helix motif
- Homeodomain
- Zinc fingers
- Leucine zipper domain
- Helix-loop-helix domain
- DNA-binding domains have characteristic consensus amino acid
sequences
- Many TFs bind in the form of homodimers (two identical protein
molecules) or heterodimers (two different molecules)
- Amino acids of TF domain enables it to very specifically recognise and
bind to nucleotides of a specific DNA element
, Helix-turn-helix motif
- Is one of the most common structural domains (e.g. homeotic/Hox TFs)
- Consists of two alpha-helices connected by a short chain of amino
acids that forms a beta sheet turn
- The two helices are held at a fixed angle
- One alpha helix is involved in nucleotide-specific recognition - called
the recognition helix
- This lies across the major groove of DNA and directly contacts
nucleotides
- Characteristics
- Other helix helps position the recognition helix correctly and
stabilises it
- TFs with this domain bind to DNA as homodimers
- Additional motifs (e.g. alpha helix) or regulatory domains may be
present on the protein
Homeotic TFs
- Have a homeodomain with helix-turn-helix motifs
- Homeotic genes encode homeotic TFs
- Homeobox is a coding nt sequence within the homeotic genes
- This encodes the homeodomain as part of the protein
