Transcription Activators in Eukaryotes

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Transcription Activators in Eukaryotes
Chapter 12

• jiacheng lin
• 2006.10.25

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• We have learned about how the RNA polymerases,
  their promoters, and the general transcription
  factors work. However, it is clear that this is
  not the whole story. The general transcription
  factors are capable of sponsoring only a very low
  level of transcription.

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• Eukaryotic cells have additional, gene-specific
  transcription factors (activators) that bind to
  DNA elements called enhancers, which could help
  transcription rise above basic level. The
  transcription activators also permits cells to
  control the expression of their genes.

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Transcription Activators in Eukaryotes

• Categories of Activators
• Structures of the DNA-binding motifs of
  Activators
• Independence of the Domains of Activators
• Functions of Activators
• Interaction among Activators
• Regulation of Transcription Factors

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Transcription activator domain structure

• DNA-binding domains
• Transcription-Activating Domains
• Dimerization domains

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DNA-binding domains

• DNA-binding domains contain motifs such as
• Zinc modules
• Homeodomain
• bHLH or bZIP motifs

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Transcription-Activating Domains

• Transcription-Activating Domains can be
• Acidic
• Glutamine-rich
• Proline-rich

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Zinc fingers

• Zinc fingers are composed of an antiparallel
  ß-strand, followed by an a-helix. The ß-strand
  contains two cysteines, and the a-helix two
  histidines, that are coordinated to a zinc ion.
  This coordination of amino acids to the metal
  helps form the finger-shaped structure.

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Zinc fingers
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Interaction with DNA

• Binding between each finger and its DNA-binding
  site relies on interactions between amino acid in
  a-helix and base in the major groove of the DNA,

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The GAL4 protein

• The GAL4 protein is a yeast activator.
• It controls a set of genes responsible for
  metabolism of galactose
• Each of the GAL4-responsive genes contains a GAL4
  target site upstream of the transcription start
  site. (UAS)

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The GAL4 protein structure

• One end of each monomer contains a DNA-binding
  motif containing six cysteines that complex two
  zinc ions. These motif also contains a short
  a-helix that protrudes into the DNA major groove
  and makes specific interactions there.
• The other end of each monomer is an a-helix which
  could forms a parallel coiled coil as it
  interacts with the a-helix on the other GAL4
  monomer.

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Are the DNA-binding domains and
transcription-activating domains independent
modules?
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The idea of Brent and Ptashne

• A hybrid protein with transcription-activating
  domains of one protein and DNA-binding domains of
  another could functions as an activator.

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summary

• The DNA-binding and transcription-activating
  domains of activator proteins are independent
  modules!

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Functions of activators

• The eukaryotic activators stimulate binding of
  general transcription factors and RNA polymerase
  to a promoter

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Two hypotheses

• The general transcription factors cause a
  stepwise build-up of a preinitiation complex
• The general transcription factors and other
  proteins are already bound to the polymerase in a
  complex, the factors and polymerase are recruited
  together to the promoter.

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• It appears that direct contacts between general
  transcription factors and activators are
  necessary. Which factors do the activators
  contact?

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Reruitment of TFIID

• A series of experiments was performed to
  identify the factor that binds to the acidic
  transcription-activating domain of the
  herpesvirus transcription factor VP16 by Stringer
  and colleagues

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Hela cell nuclear extract

• Pour through the columns

Contain protein A
Contain protein A/VP16 fusion protein
Flow-throught
Flow-throught
Run-off transcription
Run-off transcription
Support transcription
Not Support transcription
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Run-off transcription
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What is the factor binding to the
affinity column? How to identify it?
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• Stringer and colleagues knew that TFIID was
  rate-limiting for transcription in their in vitro
  system, so they suspected that was TFIID. And
  they prove it throught this way

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• Deplete a nuclear extract of TFIID by heating
  add back the material that bound to
  either the protein A column or the column
  containing the protein A/VP16-activating domain
  run off transcription

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Result
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summary

• The acidic transcription-activating domain of
  the herpesvirus transcription factor VP16 binds
  to TFIID under affinity chromatography conditions.

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• Now the peptide fingerprinting maybe more easier
  to identify the protein TFIID ?

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Recruitment of TFIIB

• Lin and Green performed their first set of
  experiments using scheme as follows

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• These results sugested that GAL4 acts by
  stimulating assembly of a preinitiation complex,
  presumably by recruiting one or more general
  transcription factors to the promoter. But which
  factor ?

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• ConclusionGAL4 help TFIIB binding
• Do TFIIB binding needs other factors?

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• Conclusion Binding of TFIIB can occur only if
  TFIID is already bound.

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summary

• Some activators with acidic activation domains,
  such as GAL4, appear to stimulate transcription
  by facilitating the binding of TFIIB to the
  preinitiation complex

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Recruitment of the holoenzyme?

• It also possible that activators recruit the
  holoenzyme as a unit.

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Evidence

• The scientist isolated a yeast mutant with a
  point mutation that changed a single amino acid
  in a holoenzyme protein(GAL11). It responded
  strongly to weak mutant versions of the activator
  GAL4. This protein binds to a region of the
  dimerization domain of GAL4(between amino acid 58
  and 97).

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• To test the hypothesis that region of GAL4
  between AA58-97 is responsible for activation by
  GAL11P, they made a plasmid encoding fusion
  protein containing the region AA58-97 of GAL4 and
  LexA DNA-binding domain.

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• Their thought if activation is really due to
  interaction between fusion protein and GAL11P
  fusing the LexA DNA binding domain to GAL11 would
  also cause activation

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• The simplest explanation for these data is that
  activation, at least in this system, can operate
  by recruitment of the holoenzyme, rather than by
  recruitment of individual general transcription
  factors.

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Is that true?

• Some studies showed that one part of the
  holoenzyme, mediator, binds to the promoter
  earlier in G1 phase than does RNA polymerase II.

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Is that true?

• Roger and colleagues reasoned that, if the
  holoenzyme binds as a unit, all of the components
  of the holoenzyme are in roughly equal amounts in
  cells.

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Attach TAP tag to the genes encoding seven
different components of the polymerase II
holoenzyme
Dot blot cell extracts from the yeast
strains Carry genes for TAP-tagged proteins
Probe the blots with an antiperoxidase antibody
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• Conclusion RNA polymerase was more abundant than
  any of the other factors, the four other general
  transcription factors were more abundant than
  either mediator or TFIIH

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• Whats your opinion?Holoenzyme or not?
• The truth may be a combination of the two
  hypotheses.

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summary

• Activation, at least in certain promoters in
  yeast, appears to function by recruitment of the
  holoenzyme, rather than by recruitment of
  individual components of the holoenzyme. However,
  other evidence suggests that recruitment of the
  holoenzyme as a unit is not common.

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thank you!