Function of Activation Domains

1
Function of Activation Domains

• Function in recruitment of components of the
  pre-initiation complex in eukaryotes (the RNAP
  holoenzyme is recruited in prokaryotes)
• Act independent of DNA-binding domains
• Can make chimeric factors that function --- i.e.,
  combine the DNA-binding domain from one factor
  and activation domain from another and get the
  expected activity

2
What basal factors are specifically recruited by
transcriptional activators such as Gal4?
3
Affinity chromatography assay for effect of
factors on transcription.
DNA construct has a GAL4 binding site upstream of
a minimal Class II promoter. It is immobilized
on an agarose bead. Proteins are added and then
the column can be washed, other proteins added,
etc.. In step (d), transcription was allowed to
proceed on the bead, and the RNA product was
analyzed by primer extension (step e).
Fig. 12.19
4
The Affinity chromatography assay shows that Gal4
promotes tight binding of TFIIB.
Factors in first incubation
TFIIB does not bind very tightly without Gal4.
From Fig. 12.21
5
GAL4 (which binds to an upstream element)

• Promotes binding of TFIIB (which promotes
  recruitment of the other factors and RNAP II).
• Probably binds directly to TFIIB (i.e., it
  doesnt work by stimulating TFIID to bind TFIIB
  tighter)
• Further work has shown that GAL4 also promotes
  assembly of the downstream factors TFIIFRNAP
  II, or TFIIE.

6
Activation from a Distance Enhancers

• There are at least 3 possible models
• Factor binding to the enhancer induces
• supercoiling
• sliding
• looping

7
Models for enhancer function
RNAP II
Basal factors
promoter
Enhancer with bound protein
Modified Fig. 12.27
8
E- enhancer Psi40- rRNA promoter
Transcription of DNAs 1-5 was tested in Xenopus
oocytes. Results good transcription from 2, 3,
and 4 (also 2 gt3 or 4) but not 5. Conclusion
Enhancer does not have to be on same DNA
molecule, but must be close. Rules out the
sliding and supercoiling models.
From Fig. 12.29
9
Looping out by a prokaryotic enhancer binding
protein visualized by electron microscopy.
NtrC protein that binds enhancer and RNAP s 54
polymerase RNAP with the 54 kDa sigma factor
10
Combinatorial Transcriptionexpression/regulation
depends on the combination of elements in the
promoter
Human Metallothionine promoter
GC box MRE- metal response element BLE- enhancer
that responds to activator AP1 GRE-
Glucocorticoid response element
Fig. 12.30