9th Feb - Co-regulator recruitment to TFs

Question 1

Which region of the NR is very sensitive to heat?

Answer 1

The LBD, it unfolds at about 46 degrees C

Question 2

How is the thermostability of the LBD improved?

Answer 2

By addition of ligand

Question 3

What do mutated V290, P467 and A233 in PPARgamma cause?

Answer 3

Very severe diabetes

Question 4

Describe the ligand binding of PPARgamma

Answer 4

H12 acts like a gate, trapping the ligand in the pocket

So that the ligand can’t leave without conformational change

Question 5

What affect did V290, P467 and A233 mutations have on the melting temperature of PPARgamma?

Answer 5

It meant that upon ligand binding the melting temperature did not alter anymore

Question 6

Outline the structure of the LBD

Answer 6

Upper portion

• -Tightly packed 3 layer helical sandwich
• -Forms rigid support for the apo-LBD

Helix 12

• -Short C-term helix of LBD
• -Fast segmental mobility
• -Has dynamic behaviour sensitive to different classes of ligand

Lower Portion
–Helices framing the ligand binding cavity

Question 7

What is the effect of ligand binding on the NR?

Answer 7

Completes the core of the receptor
Class 1 receptors are released from Hsp90 and are therefore imported into the nucleus
The global fold is stabilisied - particularly helices 3 and 11
Promote dimerisation and DNA binding
Stabilise the position of helix 12

Question 8

What are the three main methods in which co-regulators can be identified?

Answer 8

Pulldown assays

Y2H screens

Reporter Assays

Question 9

Give an example of a co-activator

Answer 9

RIP140
SRC1a
TIF2
TIF1
CBP
p300

Question 10

What is the common motif of co-activator interaction?

Answer 10

LxxLL

Question 11

Give an example of a co-repressor

Answer 11

SMRT and NCoR

Question 12

What does the co-activator require to bind to the NR?

Answer 12

Helix 12 must be active

the CoA interacts with a charge clamp - Glu on H12 and K on H3

Question 13

How do CoRs repress transcription?

Answer 13

Prevent H12 from adopting its active site configuration

H12 blocks the CoA binding position

Question 14

What is the function of Reverb?

Answer 14

Regulates circadian rhythm

Question 15

Why can’t reverb bind a CoA?

Answer 15

It has no H12

Question 16

What are SNRMs?

Answer 16

Selective Nuclear Receptor Modulators

Question 17

What is the hypothesis behind SNRMs?

Answer 17

That specific agonists can selectively target certain genes, or sets of genes, through ligands having distinct effects on different promoters

Question 18

What is the evidence supporting the SNRM hypothesis?

Answer 18

Tamoxifen has different transcriptional consequences in breast and uterine tissue

Question 19

Propose a test for the SNRM hypothesis and its fundamental problem and give an alternative test

Answer 19

Elucidate the structure of full length receptors on different promoters, but there is an unstructured hinge

Thus can compare differences in co-regulator expression in different tissues