Transcription Factors and Gene Expression

Question 1

eukaryotic gene expression and regulation at the transcriptional level requires —

Answer 1

trans-acting TFs that recognize cis-acting promoter elements through sequence specific protein-DNA and protein-protein interactions

Question 2

how can you characterize structure and function of TFs?

Answer 2

1. biochemical purification
2. expression cloning (yeast assays)
3. genome data and computational analysis

Question 3

What are the 2 things that TFs do?

Answer 3

1. bind (directly or indirectly) to DNA, sequence specifically
2. activate transcription

Question 4

What are the two domains of TFs?

Answer 4

1. DBD: sequence specific, direct contact with DNA that brinfs AD close to promoter
2. AD: direct or indirect interaction with targets affect the efficiency of transcription initiation and activity

Question 5

most TFs have several — — that each perform distinct and independent functions

Answer 5

modular domains

Ex dimerization domain, DBD, ligand binding site, transactivation domain

Question 6

What is the affect of TFs that are dimers? How do homodimers bind to DNA?

Answer 6

• enables cooperative binding, high specificity and selectivity, and combinatorial control
• binding sites for homodimers are typically palindromic (inverted repeat)

Question 7

What does it mean for a TF to bind coopertatively/combinatorially?

Answer 7

• multiple TFs interact to stabilize its binding to the DNA

Question 8

Dimerization and DNA-binding specificities can be used to group transcription factors into ‘—-’.

Answer 8

families

Ex. steroid and nuclear hormone receptor superfamilies

Question 9

What is Gal4?

Answer 9

an example of a transcription factor (activator) gene that has:
* a DBD that binds to UAS(GAL) on the target
* an AD that stimulates transcription

in yeast

deletional analysis indicates that DBD is required first for AD to function

Question 10

How to the DBD and AD compare?

Answer 10

1. they are interchangable (the AD of one TF can be swapped for the AD of another and still have activation
2. functionally independent (DBD can bind without AD present)

Question 11

What is a co-activator?

Answer 11

transactivator that doesn’t directly bind to DNA and requires protein-protein interaction with another TF to act on DNA

Question 12

How does a one-hybrid yeast screen work?

Answer 12

construct a cDNA library (plasmids each with a different target fragment of DNA)
fuse cDNA plasmid to the Gal4 AD

Question 13

How does a two-hybrid yeast screen work?

Answer 13

characterize protein-protein interactions
- bait bound to DBD; prey bound to AD
- DBD bound to promoter
- if bait and prey interact, AD is in proximity to activate transcription

Question 14

What factors increase specificity of protein-DNA interactions?

Answer 14

• specific vs non-specific contact
• length of target site (longer now always better)
• monomeric vs multimeric (dimer doubles specificity)

Question 15

What are the 4 classifications of TF by DNA-binding?

what structure are DNA-binding proteins usually?

Answer 15

1. zinc fingers
2. helix-turn-helix
3. leucine zipper
4. helix-loop-helix

DNA-binding proteins are usually alpha helices

Question 16

why are DNA-binding proteins usually alpha helices?

Answer 16

side chains point out and bind to major groove of double helical DNA

Question 17

What does the helix-turn-helix motif look like in DNA-binding TFs?

Answer 17

• 2 a-helices joined by a short strand of AAs
• fits in major groove of B-DNA
• structural and chemical complementarity between protein and DNA (HB, van der waals,

Question 18

What does the leucine zippers look like in DNA-binding TFs?

Answer 18

• extended a-helices where every 7th aa is a leucine
• creates a non polar face that is good for dimerization with another of the same protein via a coiled-coil motif
• N-terminal end has a basic aa that interacts with major groove of DNA

basic zipper is similar, but with other NP aas

Question 19

What does the basic helix-loop-helix motif look like in DNA-binding TFs?

Answer 19

• similar to basic zipper protein
• has a loop between DNA recognition helic and coiled-coil region
• usually forms heterodimers

binds DNA as a parallel, left-handed, four-helix bundle

Question 20

What does the zinc-finger motif look like in DNA-binding TFs?

Answer 20

• most common DNA-binding motif for TFs in humans and animals
• two types
• B-sheets, a-helix, both interact with zinc ion

Question 21

what are the 2 types of zinc fingers?

Answer 21

C2H2: monomeric
* 2 cysteines and 2 histidines bind to zinc ion and a-helix bainds to major groove

C4: dimeric
* nuclear receptors that bind steroid hormones
* 4 cystein residues

Question 22

What are Homeodomain-containing proteins? Structure and function

Answer 22

TFs that regulate cell development, differentiation, and
proliferation
- 3 alpha helices
- helices 2 and 3 look like helix turn helix
- helix 3 connects with major groove (recognizes DNA)
- helix 1 also connects with a different major groove

stable interaction with 3 helices

Question 23

ADs are different in structure to DBDs because

Answer 23

usually exist as unfolded motifs

structure and function are not clearly related

Question 24

What are the 3 types of activation domains?

Answer 24

1. acidic: amphipathic helix with acidic residues on one face (Gal4)
2. Gln-rich
3. Pro-rich