Lecture 3, Fall 2004

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Lecture 3, Fall 2004 Transcription activation
domains and their mechanism of action.
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Transcription activation domains. The domain swap
and trans-activation assay have resulted in the
identification of several distinct activation
domains.
Often, in contrast to the above figure,
investigators fuse putative activation domains to
the Gal4-DBD.
In the case of a Gal4-fusion, plasmid 1 would
encode the fusion protein and plasmid 2 would
have binding sites for Gal4-DBD.
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Many mammalian activation domains are enriched in
particular amino acids.
Gal4-VP16 is the most widely used activator in
the transcription field, yet it does not exist in
nature.

• Examples
• acidic - VP16 from from herpes virus
• glutamine rich - Sp1 from mammals
• proline rich
• serine/threonine rich

Yeast appear to only have acidic activation
domains. The implication of this is that there
are activation mechanisms found in higher
eucaryotes that arent found in yeast.
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To understand the mechanism by which activation
domains activate transcription, investigators
have sought to identify what these domains
interact with.
GST pull-down assay

• Different types of Pull-down assays
• Purify tagged protein from E.coli, immobilize,
  incubate with eucaryotic cell extract.
• Express tagged protein in eucaryotic cell and see
  what copurifies.
• Purify tagged protein and putative partner from
  separate E.coli strains, immobilize tagged
  protein and determine if putative partner can
  bind.

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Co-immunoprecipitation experiments.
Epitope tags are selected based on commercially
available antibodies eg. anti-FLAG, anti-HA.
Protein A or Protein G are commonly used in
immunoprecipitations because they bind the
constant region of many antibodies. Hence
protein A or protein G sepharose is often used in
I.P.s to pull-down antibody-antigen
complexes.
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• Protein interaction studies have identified many
  potential targets for activation domains.
• The acidic VP16 activation domain interacts with
  TBP, TAF40, TFIIB, TFIIH, chromatin remodeling
  proteins, histone acetyltransferase.
• The glutamine-rich Sp1 activation domain
  interacts with TAF2 in mammals and Drosophila.

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Recruitment of factors to promoters by
sequence-specific DNA binding proteins appears to
be a common mechanism of activation.
Some evidence 1. Activator bypass. 2. Yeast
2-hybrid assay.
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Hypothesis
Activator bypass
Engineered so DNA binding domain is fused to
subunit of mediator.
The activation domain has been removed, and the
DBD is fused directly to the putative target.
Strong activation is observed.
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This can be provided as a library to screen for
unknown proteins that interact with a known bait.
Yeast 2-hybrid screen.
The interactions are occurring in live yeast
cells to induce expression of the reporter. Some
researchers consider this an in vivo assay.
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Activators often require Coactivators that lack
DNA binding domains to function. By definition,
coactivators do not bind directly to specific DNA
sequences.

• Coactivator is often operationally defined - it
  depends on how the experiment was set up.
• TAFs were considered co-activators because they
  had to be added to an in vitro transcription
  system containing isolated TBP in order to
  reconstitute activator-dependent transcription.
• Currently, coactivator usually refers to a
  histone acetyltransferase, chromatin remodeling
  factor, or mediator.