Lecture 4-Transcription II (Martin)

Question 1

What is the difference between genes with the TATA/CCAAT boxes and those for GC boxes with respect to transcription initiation?

Answer 1

• TATA/CCAAT boxes: TATA=-25/30bps; CCAAT box=-70–90bp. Usually only have one transcription start site. TFs will usually bind the TATA box and recruit pol
• GC boxes: TATA/CCAAT boxes absent. Rich in Sp1 binding sites (a ubiquitous TF) and usually has multiple transcription start sites

Question 2

What is the order in which PIC proteins bind? Explain the functions of each if we know it.

Answer 2

• IID: has a TATA binding protein
• IIB
• IIA: stabilizes IID interaction with TATA
• IIF/Pol II: IIF keeps Pol II from inappropriately binding and transcribing DNA and binds IIB to bring Pol to PIC
• IIE: unwinds DNA
• IIH: unwinds DNA and phosphorylates Pol II so it can recruit proofreading machinery
• IIJ

Question 3

Actinomycin D and Acridine

Answer 3

• non-specific inhibitors that intercalate between GC residues to prevent transcription

Question 4

rifampicin

Answer 4

binds the beta subunit of and inhibits the prokaryotic Pol

Question 5

One of the functions of CCAAT box?

Answer 5

• to determine which genes are on/off

Question 6

Once the PIC begins transcription what is released? Why?

Answer 6

• IIF

- so elongation factors can bind

Question 7

TFs are not part of ______ and can act as either _____ or ______

Answer 7

• transcriptional machinery

- enhancers or repressors

Question 8

Sp1

Answer 8

TF that is constitutively active in cells and binds GC boxs’ Sp1 binding sites

Question 9

enhancers do what? (3)

Answer 9

• increase the rate that Pol binds
• enhances gene expression
• can act independently of location and orientation

Question 10

Where can enhancers not be?

Answer 10

• very close to the transcription start site–this implies the importance of looping DNA

Question 11

TFs usually function in what conformation?

Answer 11

dimers

Question 12

What are the functions of TFs?

Answer 12

• recruit repressors/corepressors
• recruit activators/coactivators
• interact with PIC and coregulators

Question 13

Function of a TF can depend on ______. Ex?

Answer 13

What other proteins it binds
- ex: myc will usually bind mad which binds max and turns genes off but can also turn on genes to make cells grow inappropriately

Question 14

GR uses what kind of activation?

Answer 14

• ligand binding

Question 15

DNA binding regions of proteins tend to be ______. Why?

Answer 15

• basic

- the positive charge keeps them bound to DNA

Question 16

Why are Leu zippers called such?

Answer 16

• they have Leu’s every 7th aa that cause hydrophobic binding of the 2 alpha helices of the protein

Question 17

A binding motif very similar to Leu zippers is _______.

Answer 17

Helix-loop-helix

Question 18

GR has what type of DNA binding structure?

Answer 18

• Zn finger

Question 19

Describe Zn finger structure

Answer 19

• either 4 Cys or 2 His/2 Cys surrounding a Zn

Question 20

Do all Zn fingers confer binding specificity?

Answer 20

• no

Question 21

TFs that interact with PIC generally have what 3 characteristics?

Answer 21

• acidic (negative charge)
• Pro-rich
• Gln-rich

Question 22

GAL4 enhancer protein uses what kind of domain to bind TFs?

Answer 22

• acidic

Question 23

Describe hypersensitive sites?

Answer 23

usually contain the promoters of genes but also have regulatory protein binding sites so its thought that regulatory binding at these disrupts the nucleosomal activity

Question 24

Histone tails have what property?

Answer 24

• very basic, therefore + charge

Question 25

coactivators have _______ activity.

Answer 25

acetylase

Question 26

activators functions (2)?

Answer 26

• open up histone complex

- aid in TF binding

Question 27

Point mutations in the locus control region of the globin operon results in _______(3)

Answer 27

• loose TF binding
• inability of chromatin to decondense because of it
• enhancers can’t help

Question 28

CpG islands common in ______

Answer 28

promoters

Question 29

most of the proteins that turn of genes do so how?

Answer 29

• binding of deacetylases to methylated CG repeats

- some that bind the methylated CGs recruit others to deacetylate

Question 30

Actinomycin/Acridin (2)

Answer 30

• intercalating agents that prevent transcription

- intercalate between CGs

Question 31

rifampicin

Answer 31

• binds beta subunit of prokaryotic pol to inhibit transcription

Question 32

alpha-amanitin

Answer 32

Blocks eukaryotic Pol II (pol III at high doses)