CMB2001/L02 Activators

Question 1

What 3 elements promote efficiency of transcription?

Answer 1

Core promoter element
Initiator
Activator binding sites

Question 2

What is the function of UAS/enhancer elements?

Answer 2

Activate transcription (from basal level) for higher efficiency

Question 3

What is the role of a factor in a common sequence element?

Answer 3

Aids binding of common sequence elements to transcription factors/activators

Question 4

What is the sequence and factor associated with the GC box?

Answer 4

GGGCGG
Sp1

Question 5

What is the sequence and factor associated with the octamer?

Answer 5

ATTTGCAT
Oct-1

Question 6

What is the sequence and factor associated with the CAAT box?

Answer 6

GGCCAATCT
NFY

Question 7

Where are common sequence elements often located?

Answer 7

Close to core promoter

Question 8

When do common sequence elements become active?

Answer 8

They are constitutively active (continually)

Question 9

Name 2 response elements.

Answer 9

SRE
HSE

Question 10

What induces and binds SRE?

Answer 10

Induced by growth factors
Binds serum response factor (SRF)

Question 11

What induces and binds HSE?

Answer 11

Induced by heat shock
Binds heat shock factor

Question 12

Describe combinatorial control of transcription.

Answer 12

The type and combination of elements dictates when and at what level a gene is transcribed

Question 13

What location and orientation do enhancers work in?

Answer 13

All locations/orientations

Question 14

How are activators associated with enhancers/UAS enhancers brought into contact with GTF/DNA poll II at the core promoter?

Answer 14

By looping out of the intervening DNA

Question 15

How many DNA binding domains and activation domains are on a strand of DNA?

Answer 15

1 DNA binding domain
Maybe more than 1 activation domain

Question 16

Give 2 DNA binding domains.

Answer 16

Leucine zipper
Homeodomain
Helix loop helix
Zinc finger

Question 17

How are activation domains often characterised?

Answer 17

According to amino acid composition

Question 18

Give 3 features of activation domains.

Answer 18

Lack of sequence conservation and structural information
Generally thought to be unstructured
Contain multiple short segments that work together in additive fashion
Interact with other proteins in transcriptional machinery

Question 19

How do activation domains promote transcription? (2)

Answer 19

Recruit transcription factors and coactivators
Modify chromatin structure to make DNA more accessible for transcription

Question 20

How are activators analysed in vitro by gel shift/electrophoretic mobility shift assays?

Answer 20

Activator and radiolabelled probe DNA run on non-denaturing acrylamide gel
Gel separates based on size and mass
Only measure ability to bind DNA, not transcription

Question 21

How are activators analysed in vitro by transcription assays? (2)

Answer 21

RNA pol II, GTFs, DNA template, radiolabelled rNTPs assayed
Requires activator to have functional DNA binding domain and activation domain

Question 22

How are activators analysed in vivo? (3)

Answer 22

Reporter assays
Plasmid 1 encodes protein X
Reporter gene of plasmid 2 activated by protein X binding
Reporter gene transcripts produced

Question 23

Describe the 5 steps of chromatin immunoprecipitation.

Answer 23

Crosslink bound proteins to DNA
Isolate chromatin and shear DNA
Precipitate chromatin with protein-specific antibody
Reverse cross-link and digest protein
Analyse DNA using PCR or sequencing

Question 24

Give 4 ways in which activators work.

Answer 24

Cooperative binding- promote binding of additional activator (uncommon)
Stimulate complex assembly (major)
Release stalled RNA pol (stimulate activity)
Modulation of chromatin

Question 25

Which components of the PIC complex do activators interact with to promote assembly? (3)

Answer 25

TFIID (via TAFs)
TFIIB
Mediator

Question 26

What is the mediator complex?

Answer 26

An additional factor required for transcription activation in minimal in vitro transcription systems

Question 27

Describe the structure of mediator. (3)

Answer 27

22 polypeptides
Can exist on its own or associated with RNA pol II through C-terminal domain
Composed of 3 domains - head, middle, tail

Question 28

Describe the function of mediator. (2)

Answer 28

Provides a bridge between activators and RNA pol II
Mediator-activator interactions aid recruitment of RNA pol II to enhance PIC formation

Question 29

Where can RNA pol II stall?

Answer 29

On or near the promoter

Question 30

Give an example of an activator releasing stalled RNA pol. (3)

Answer 30

Heat shock genes e.g., hsp70
IN absence of heat shock, RNA pol II pauses after -50nts
Heat shock activates HSF transcription factor which interacts with RNA pol II
Releases it from the pause