Transcription Factors Flashcards
How do transcription factors bind to DNA?
Originally it was thought the DNA needed to be open up for transcription factors to access the DNA
The outside of the DNA double helix can be “read” by proteins
DNA binding proteins “see” sequence information from the outside of the double helix
It binds in the major groove
More sequence information is present in the major groove than the minor groove = more DNA binding proteins recognising the major groove
How do the transcription factors bind the DNA?
Different amino acids hydrogen bond with bases in the major or minor groove
Typically, protein/DNA interface has 10-20 contacts of the type shown
Each contribute to the strength of the DNA/protein interaction
They also contribute to the specificity - in the Nano molar range
The protein often presents an alpha helix to form these hydrogen bond interactions with the major groove
What are the different types of trancription factor categories that can bind?
Helix-turn-helix
Zinc fingers
Leucine zippers
Helix-loop-helix
Describe the Helix-turn-Helix DNA binding motif?
C-terminal - DNA recognition helix binds in major groove
N-Terminal helix - helps to position the recognition helix
(two helices in prokaryotes)
They often bind as dimers
Examples - tryptophan repressor, lambda Cro, CAP fragment
The binding half-sites are separated by exactly 1 turn of the double helix
Doubling the number of contacts, squares the affinity constant
Which proteins always contain Helix-turn-Helix, within the same structural context?
Homeodomain proteins
60 amino acid domain found in all homeotic proteins - with 3 helices (in eukaryotes)
Helices 1 and 2 provide structural context
Helix 3 is a recognition helix
N-terminal extension (preceding helix 1) binds (hooks) in minor groove
This stabilises the DNA binding motif
1 single amino acid change in the 60 aa, can alter where the domain binds to in the DNA
Describe the Zinc finger DNA binding domains?
Binds to major groove in DNA
Two main types:
Cys2-Cys2: Adenovirus E1A, yeast GAL4, steroid receptors
AND
Cys2-His2: Sp1, TFIIIA, Drosophila Kruppel
This can’t be mutated into Cys-Cys type
Zinc coordinates four amino acids to hold one end of the a-helix firmly to one end of the b-sheet
Describe the Cys2-His2 Zinc fingers?
They can occur in tandem
This allows an almost continuous stretch of a-helices that can contact the major groove
Transcription factors do not have to use all their Zn fingers – e.g. CTCF 10 Zn fingers or Ikaros family ~4 Zn fingers (depending on the splice variants)
Describe the Cy2-Cy2 Zinc fingers?
They form dimers
The two halves of the recognition site are separated by 1 turn of the DNA helix
The N-terminal Zinc finger = binding DNA
The C-terminal Zinc finger = dimerisation
Evidence - Changing 2 amino acids out 777 can completely alter the DNA binding specificity
The 2nd zinc finger determines the optimal spacing between the two halves of the palindromic recognition site
Examples: Nuclear hormone receptors
Describe steroid receptors?
They function as dimers - recognising palindromic sites
They have common domains
Only the spacing between the two is changing between the different types
The domains can be swapped to change the binding of the hormone receptors i.e. genes normally induced by glucocorticoid are induced by estrogen - if the domains are swapped
Describe Leucine Zippers?
You must form a dimer between two monomers of the protein = create the DNA binding domain
Leucine Zippers bind as dimers with a-helices contacting DNA sequences in the major groove
Leucine is repeated every 7 amino acids, allowing dimerisation of the a-helices via hydrophobic interactions
This generates an inverted Y-type structure that grips the DNA like a clothes peg
What can be changed within Leucine Zippers?
Heterodimerisation of Leucine Zipper proteins can alter their DNA binding specificity
Dimers of different factors can recognise the same sequence but have different effects
E.g. Myc:max is an activator while Mad:max is a repressor
The heterodimer recognises different DNA half sites compared to the homodimer
Describe the Helix-Loop-Helix motif?
This motif mediates dimerisation and DNA binding
Short helix - mediates dimerisation
Separated by a loop from a long helix that mediates DNA binding
Dimerisation regulates DNA binding
What can happen to Helix-Loop-Helix proteins?
They can be inactivated by dimerisation with partners that lack DNA binding domains
If a monomer lacks a functional DNA binding domain - this prevents the HLH protein from binding to DNA
The repressor protein can be expressed at different levels - allowing control
What are designer transcription factors? Examples?
We can design them so we can choose where they bind
E.g. Zinc finger nucleases and TALE system
Describe Zinc finger nucleases?
Can be hooked to Fok I endonuclease to introduce breaks at specific sites in the genome
Disadvantages
Each finger recognises a triplet of bases; not all triplets are recognised specifically - we would need 64 Zinc fingers
For Fok1 cutting, proteins must bind palindromic DNA sequences as a dimer (spacer 5=7 bp)
