Specific Transcription Factors Flashcards
How to TFs bind to specific DNA sequences?
through their DNA binding domains
How do TFs interact with the basal transcription machinery?
through protein-protein interaction via their activation domains
What are the 2 techniques used to identify the binding site of DNA and TFs?
- DNase I foot printing
- EMSA
What part of DNA do TFs make the most contact with?
the major groove
How does DNA interact with proteins?
- H bonds
- ionic bonds
- hydrophobic interactions
What does Asn have?
a side chain that directly interacts with the major groove of the DNA molecule
How are TFs classified?
based on their conserved DNA binding domains
What are the 4 main TF binding domains?
- zinc finger
- homeodomain
- helix-loop-helix
- leucine zipper
What does each TF control?
multiple genes
What is a typical zinc finger?
~23 amino acids with 2 cysteines and 2 histidines that chelate a zinc atom
How is the zinc finger domain formed?
by the interaction between the zinc atom with an alpha helix and an antiparallel beta sheet
What does each zinc finger recognise?
3 GC-rich nucleotides
What part of the zinc finger recognises the nucleotides?
amino acids 1, 2, 3 and 6
How can zinc fingers be artificially designed?
using ZFN (zinc finger nuclease) for genome editing
What is the homeodomain?
a conserved 60 amino acid domain particularly important during development
How is the homeodomain folded?
into 3 alpha helices (helices 2 and 3 are similar to the HTH motif)
What parts of the homeodomain contact the major and minor groove of DNA?
- N-terminal arm lies in minor groove
- helices 1 and 2 lie above the DNA
- helix 3 lies in the major groove
What is the recognition sequence of homeodomain?
a core ATTA
What are the 2 binding domains that some homeodomain proteins contain?
- POU domain that cooperates with the homeodomain to increase the binding specificity and affinity
- paired domain binds to target DNA independent of the homeodomain
What is the bHLH?
a basic domain for DNA binding made up of 2 unequal helices
What allows flexibility of the bHLH?
a long loop which allows the helices to fold over each other and form a dimer through the HLH domain
What are bHLH proteins involved in?
cell differentiation in eukaryotes
What are the 3 bHLH proteins?
- MyoD
- E2A
- Id
What is MyoD?
a bHLH protein specifically expressed in muscle cells
What is Id?
a bHLH protein without a basic region that forms a dimer with MyoD that inhibits formation of the MyoD-E2A dimer
What is the E box?
the DNA binding motif for bHLH proteins; CACGTG
What happens in the presence of Id?
the E box is not required and MyoD-E2A dimers do not form
What is the frequency of leucine residues on the leucine zipper?
every 7 amino acids on only one side of the helix
What is the leucine zipper?
an amphipathic helix; one face of the helix contains hydrophilic side chains and the other face contains hydrophobic side chains
When does the leucine zipper protein function?
when it is a dimer
What do Myc and Max contain?
both bHLH and Zip domains
What do Myc and Max do?
form heterodimers by Zip domain interaction and bind to E box (CACGTG) of target genes
What is Myc?
an important TF that regulates the transcription of ~15% of cellular genes
What can overexpression of Myc cause?
cancer
What can ADs interact with?
directly with GTFs or with cofactors
What are the 4 types of activation domains?
- acidic domains
- gluatmine-rich domains
- proline-rich domains
- isoleucine-rich domains
What is acidic/9aaTAD?
a loose consensus sequence essential for transcriptional activation
What does acidic/9aaTAD interact with?
coactivators TAF9 and CBP/p300
What is the acidic AD of CREB?
a bZIP TF
How does the acidic AD of CREB work?
it folds into two amphipathic α-helices and interacts with co-activator CBP
What are nuclear receptor TFs?
a group of zinc finger proteins that bind steroids in the cytoplasm and move into the nucleus where they bind the DNA and dimerise to activate transcription
What does ligand binding to nuclear receptors cause?
- release of inhibitory proteins
- conformational change to seal the ligand
- binding of co-activator
- nuclear receptor binds to HRE to activate transcription by enhancing formation of transcription IC
What does assembly of the multi-protein complex on HRE do?
enhance transcription by interaction with GTFs, TAFs and mediator protein
What does every nuclear receptor have?
2 fingers; one for DNA binding and the other for dimerisation
What do nuclear receptors bind to?
a short 15 nucleotide palindromic DNA sequence
What are TGFs involved in?
a variety of activities in development, immune regulation, tissue repair, cancer
What are the 4 steps of TGF-β signalling?
- extracellular ligand TGFβ binding results in tetramerisation of two receptors to phosphorylate intracellular domains
- activation of R-Smad
- formation of Smad trimers with C-Smad, common Smad4
- translocation to the nucleus and binding to responsive element to activate target genes
What are the 5 steps of cAMP signalling?
- signal molecule binding to activate GPCR
- activated G protein and AC catalyse formation of cAMP
- PKA activation: cAMP binds to regulatory subunits to release catalytic subunits
- translocation to the nucleus to activate CREB by phosphorylating Ser-133
- binding to CRE and recruiting co-activator CBP activates target genes
What is canonical Wnt signalling important for?
development and cancers
What are the 5 steps of the canonical Wnt pathway?
- Wnt binds to Frazzled receptor
- Dishevelled and LRP are recruited
- inhibitory complex dissociates
- unphosphorylated β-catenin is released and translocates to the nucleus
- co-pressor Gruncho is displaced to activate target genes with TF LEF1/TCF
What does TF dimerisation do?
increase DNA binding specificity and affinity as well as functional diversity
What does each binding domain recognise?
4-6 nucleotides
How can a single TF control several genes?
by interacting with different factors
What does glucocorticoid receptor do?
co-ordinate expression of many different genes
What does the effect of GR on GR-responsive genes depend on ?
- presence of the GR
- presence of the ligand
- presence of other regulatory proteins
- binding sites on the gene
