Transcription Part 2

Question 1

What are 4 protein domains that interact with DNA?

Answer 1

Zinc Fingers, Nuclear hormone receptor DNA binding Domain, Helix-Turn-Helix, Leucine Zipper

Question 2

Differences between Gal4 and VP16 Activation Domains

Answer 2

Gal4 can bind DNA directly once activated.

VP16 does not bind directly to DNA. Has acidic C-terminal region that can interact with other DNA-binding domains.

Question 3

How does the mediator interact with RNA Pol?

Answer 3

Interacts with unphosphorylated RNA polymerase CTD (think about why this is the case)

Question 4

How is the mediator regulated?

Answer 4

CDK8 inhibits mediator activity. CDK8 has to disassociate before CTD can interact.

Question 5

Properties of Enhancers (3)

Answer 5

can enhance transcription up to 1 megabase away, can be upstream or downstream of TSS, orientation can be inverted

Question 6

How are enhancers brought in close proximity to promoters?

Answer 6

Chromatin has to bend. Cohesin can help facilitate this.

Question 7

How to identify enhancers? (4)

Answer 7

1) Map TF binding site, compare to TSS
2) Look for nuclease sensitive sites (gently digest w/ DNase)
3) Look for H3K4m1 and H3K27ac marks
4) Clone sequences that enhance txn

Question 8

Technique: ChIA-PET

Answer 8

Chromatin Interaction Analysis by Paired-End Tag Sequencing, similar to Chromosome Conformation Capture.

Look at enhancer locations

Question 9

Technique: STARR-seq

Answer 9

Self-transcribing active regulatory region sequencing
Place sequence between ORF and pA site
Only cells that have enhancer sequence will transcribe cDNA. Can characterize enhancers.

Question 10

How are super enhancers characterized?

Answer 10

Have high levels of mediator, especially MED1

Question 11

Structure: Nucleosome

Answer 11

2 copies of H2A, H2B, H3, H4

147 bp wrapped 1 3/4 turns

Question 12

How are nucleosomes arranged around genes?

Answer 12

Depleted at 5’ and 3’ ends, regularly spaced downstream of promoter.

Question 13

What is required for chromatin remodeling?

Answer 13

ATP. Be able to describe experiment that showed this

Question 14

Mechanism: Chromatin Remodeling

Answer 14

Loop model. Protein pulls DNA and creates a bubble. The histone core is pulled along DNA, shifts the bubble formed. ATP is required for this to occur.

Question 15

Two primary histone modifications + writers and ereasers

Answer 15

Methyl, methyltransferases and demethylases

Acetyl, Acetyltransferases and acetylases

Question 16

Does histone acetylation stabilize or destabilize nucleosomes and why?

Answer 16

Destabilizes nucleosomes by neutralizing positive charge of Lys (Lys can interact favorable with PDE bond of DNA). Facilitates hetero to euchromatin transition.

Question 17

Two HATs mentioned in class

Answer 17

GCN5, SAGA

Histone Acetyltransferase
SAGA maps behind promoters with ChEC-seq

Question 18

Mechanism: Sir Proteins

Answer 18

Sir 1, 3, 4 bind de-acetylated chromatin
Sir2 is deacetylase
combined action causes deacetylase spread, which shifts chromatin to heterochromatin state.

Question 19

Does methylation activate or repress genes?

Answer 19

Do both.

H3K27me turns promoters off
H3K4me turns promoters on

Question 20

Mechanism: PRC2

Answer 20

Polycomb Repressive Complex. Interacts with dinucleosomes and spreads H3K27me3 marks.

One protein binds H3K27me3, another domain, SET, can add methyl groups to adjacent nucleosome.

Question 21

How does the mediator interact with transcription factors?

Answer 21

Mediator interacts with activation domains through multiple weak interactions found in MED15.