Specific Transcription Factors

Question 1

How to TFs bind to specific DNA sequences?

Answer 1

through their DNA binding domains

Question 2

How do TFs interact with the basal transcription machinery?

Answer 2

through protein-protein interaction via their activation domains

Question 3

What are the 2 techniques used to identify the binding site of DNA and TFs?

Answer 3

• DNase I foot printing
• EMSA

Question 4

What part of DNA do TFs make the most contact with?

Answer 4

the major groove

Question 5

How does DNA interact with proteins?

Answer 5

• H bonds
• ionic bonds
• hydrophobic interactions

Question 6

What does Asn have?

Answer 6

a side chain that directly interacts with the major groove of the DNA molecule

Question 7

How are TFs classified?

Answer 7

based on their conserved DNA binding domains

Question 8

What are the 4 main TF binding domains?

Answer 8

• zinc finger
• homeodomain
• helix-loop-helix
• leucine zipper

Question 9

What does each TF control?

Answer 9

multiple genes

Question 10

What is a typical zinc finger?

Answer 10

~23 amino acids with 2 cysteines and 2 histidines that chelate a zinc atom

Question 11

How is the zinc finger domain formed?

Answer 11

by the interaction between the zinc atom with an alpha helix and an antiparallel beta sheet

Question 12

What does each zinc finger recognise?

Answer 12

3 GC-rich nucleotides

Question 13

What part of the zinc finger recognises the nucleotides?

Answer 13

amino acids 1, 2, 3 and 6

Question 14

How can zinc fingers be artificially designed?

Answer 14

using ZFN (zinc finger nuclease) for genome editing

Question 15

What is the homeodomain?

Answer 15

a conserved 60 amino acid domain particularly important during development

Question 16

How is the homeodomain folded?

Answer 16

into 3 alpha helices (helices 2 and 3 are similar to the HTH motif)

Question 17

What parts of the homeodomain contact the major and minor groove of DNA?

Answer 17

• N-terminal arm lies in minor groove
• helices 1 and 2 lie above the DNA
• helix 3 lies in the major groove

Question 18

What is the recognition sequence of homeodomain?

Answer 18

a core ATTA

Question 19

What are the 2 binding domains that some homeodomain proteins contain?

Answer 19

• POU domain that cooperates with the homeodomain to increase the binding specificity and affinity
• paired domain binds to target DNA independent of the homeodomain

Question 20

What is the bHLH?

Answer 20

a basic domain for DNA binding made up of 2 unequal helices

Question 21

What allows flexibility of the bHLH?

Answer 21

a long loop which allows the helices to fold over each other and form a dimer through the HLH domain

Question 22

What are bHLH proteins involved in?

Answer 22

cell differentiation in eukaryotes

Question 23

What are the 3 bHLH proteins?

Answer 23

• MyoD
• E2A
• Id

Question 24

What is MyoD?

Answer 24

a bHLH protein specifically expressed in muscle cells

Question 25

What is Id?

Answer 25

a bHLH protein without a basic region that forms a dimer with MyoD that inhibits formation of the MyoD-E2A dimer

Question 26

What is the E box?

Answer 26

the DNA binding motif for bHLH proteins; CACGTG

Question 27

What happens in the presence of Id?

Answer 27

the E box is not required and MyoD-E2A dimers do not form

Question 28

What is the frequency of leucine residues on the leucine zipper?

Answer 28

every 7 amino acids on only one side of the helix

Question 29

What is the leucine zipper?

Answer 29

an amphipathic helix; one face of the helix contains hydrophilic side chains and the other face contains hydrophobic side chains

Question 30

When does the leucine zipper protein function?

Answer 30

when it is a dimer

Question 31

What do Myc and Max contain?

Answer 31

both bHLH and Zip domains

Question 32

What do Myc and Max do?

Answer 32

form heterodimers by Zip domain interaction and bind to E box (CACGTG) of target genes

Question 33

What is Myc?

Answer 33

an important TF that regulates the transcription of ~15% of cellular genes

Question 34

What can overexpression of Myc cause?

Answer 34

cancer

Question 35

What can ADs interact with?

Answer 35

directly with GTFs or with cofactors

Question 36

What are the 4 types of activation domains?

Answer 36

- acidic domains - gluatmine-rich domains - proline-rich domains - isoleucine-rich domains

Question 37

What is acidic/9aaTAD?

Answer 37

a loose consensus sequence essential for transcriptional activation

Question 38

What does acidic/9aaTAD interact with?

Answer 38

coactivators TAF9 and CBP/p300

Question 39

What is the acidic AD of CREB?

Answer 39

a bZIP TF

Question 40

How does the acidic AD of CREB work?

Answer 40

it folds into two amphipathic α-helices and interacts with co-activator CBP

Question 41

What are nuclear receptor TFs?

Answer 41

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

Question 42

What does ligand binding to nuclear receptors cause?

Answer 42

1. release of inhibitory proteins 2. conformational change to seal the ligand 3. binding of co-activator 4. nuclear receptor binds to HRE to activate transcription by enhancing formation of transcription IC

Question 43

What does assembly of the multi-protein complex on HRE do?

Answer 43

enhance transcription by interaction with GTFs, TAFs and mediator protein

Question 44

What does every nuclear receptor have?

Answer 44

2 fingers; one for DNA binding and the other for dimerisation

Question 45

What do nuclear receptors bind to?

Answer 45

a short 15 nucleotide palindromic DNA sequence

Question 46

What are TGFs involved in?

Answer 46

a variety of activities in development, immune regulation, tissue repair, cancer

Question 47

What are the 4 steps of TGF-β signalling?

Answer 47

1. extracellular ligand TGFβ binding results in tetramerisation of two receptors to phosphorylate intracellular domains 2. activation of R-Smad 3. formation of Smad trimers with C-Smad, common Smad4 4. translocation to the nucleus and binding to responsive element to activate target genes

Question 48

What are the 5 steps of cAMP signalling?

Answer 48

1. signal molecule binding to activate GPCR 2. activated G protein and AC catalyse formation of cAMP 3. PKA activation: cAMP binds to regulatory subunits to release catalytic subunits 4. translocation to the nucleus to activate CREB by phosphorylating Ser-133 5. binding to CRE and recruiting co-activator CBP activates target genes

Question 49

What is canonical Wnt signalling important for?

Answer 49

development and cancers

Question 50

What are the 5 steps of the canonical Wnt pathway?

Answer 50

1. Wnt binds to Frazzled receptor 2. Dishevelled and LRP are recruited 3. inhibitory complex dissociates 4. unphosphorylated β-catenin is released and translocates to the nucleus 5. co-pressor Gruncho is displaced to activate target genes with TF LEF1/TCF

Question 51

What does TF dimerisation do?

Answer 51

increase DNA binding specificity and affinity as well as functional diversity

Question 52

What does each binding domain recognise?

Answer 52

4-6 nucleotides

Question 53

How can a single TF control several genes?

Answer 53

by interacting with different factors

Question 54

What does glucocorticoid receptor do?

Answer 54

co-ordinate expression of many different genes

Question 55

What does the effect of GR on GR-responsive genes depend on ?

Answer 55

- presence of the GR - presence of the ligand - presence of other regulatory proteins - binding sites on the gene