Activation of Eukaryote Transcription

Question 1

What are the goals of activators in terms of the PIC?

Answer 1

Increase the association constant and decrease the dissociation constant of the PIC complex
Increase stability and promoter accessibility
Binds direction to DNA sites

Question 2

Components of activators

Answer 2

Trans-activation domain
DNA binding domain
NLS (for nuclear import)

Question 3

5 ways for activators to directly act on GTFs/DNA

Answer 3

Note: alpha helix recognizes DNA major groove

1. Helix-turn-helix
2. Homeodomain
3. Zn finger domain
4. Basic helix-loop-helix
5. Leucine zipper/basic zipper

Question 4

What is the helix-turn-helix?

Answer 4

2 alpha helices (monomer)
CTD binds DNA
NTD positions CTD

Question 5

What is the homeodomain?

Answer 5

3 alpha helices (monomer)
CTD binds DNA
Other helices position CTD and bind DNA or other protein

Question 6

What is the Zn finger domain?

Answer 6

Cys/His fold around Zn ion to position alpha helix to bind DNA

Question 7

What is the basic helix-loop-helix?

Answer 7

Dimerization with 2 DNA binding motifs (NTD)
Loop separating DNA binding domain and dimerization domain
Ex: myr, mad, max

Question 8

What is the Leucine zipper/basic zipper?

Answer 8

Basic dimerization of Leu rich region with NTDs binding DNA
Ex: c-fos, c-jun, C/EBPb

Question 9

Important controls for EMSA

Answer 9

Always have excess probe
1. unlabeled specific probe (no shift)
2. labeled probe that doesn’t bind (no shift)
3. antibody specific to activator (supershift)

Question 10

Explain ChIP

Answer 10

To demonstrate activator binding to specific regulator gene
1. Formaldehyde fix proteins onto DNA/histones
2. Sonicate to break up H1 linkers
3. Antibody specific to activator
4. Immunoprecipitation (only get genes w/ activator)
5. Reverse crosslinking (everything separated)
6. DNA purification
7. PCR for genes of interest

Question 11

Important controls for ChIP

Answer 11

1. No antibody
2. Different antibody that doesn’t bind activator
3. PCR to different genes

Question 12

What is the Gal4 Hybrid Assay

Answer 12

Fusing regions of a factor to Gal4 to find activation domain (TAD) by reporter gene activation

Question 13

Describe how activators utilize DNA bending and qualities of them

Answer 13

Facilitate interactions between factors by making them closer together
Allowing enhancers to act at further distances through DNA looping and binding
Lack TAD, have DNA binding domain
Ex: HMG proteins

Question 14

Describe purpose of HMG proteins

Answer 14

Small proteins with low DNA specificity
Bind minor groove and bend DNA
Ex: TBP binding to TATA Box

Question 15

What are the 2 ways to modify chromatin structure near the core promoter

Answer 15

Histone Modification
ATP-Dependent Nucleosome Remodeling

Note: heterochromatin –> euchromatin

Question 16

Describe histone modification in terms of activators

Answer 16

Post-translational modifications of histone tails (H3/H4)
Direct = altering charges
Indirect = making binding sites for factors
By acetylation and methylation

Question 17

Results of histone acetylation

Answer 17

Neutralizes K+ which loosens the histone’s grip to DNA and increase accessibility to other factors
Allows binding of bromodomain to histone
Catalyzed by HATs

Acetylation = activation

Question 18

Results of histone methylation

Answer 18

Catalyzed by HMT to methylate heterochromatin
Can activate OR repress transcription

Question 19

Function of bromodomain of TFIID

Answer 19

Reader of histone code

Note: histone modifying enzymes = writers

Question 20

Describe ATP-dependent nucleosome remodeling

Answer 20

Using ATP to make DNA more accessible to activators/PIC
Recruited by other activators already on DNA
Have remodeling complexes

Question 21

Describe remodeling complexes in ATP-dependent nucleosome remodeling

Answer 21

ATPase with accessory functions
Goal is to unwrap, shift, and conformationally change nucleosomes to facilitate activator/PIC binding