A transcription reinitiation intermediate that is stabilized by activator

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A transcription reinitiation intermediate that is
stabilized by activator
Presentation by Castor Phan Huilin Koh
RNA Polymerase II
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How does RNA Pol II begin working?
INITIATION of RNA Pol II begins by assembly of
transcription factors on ssDNA or nicked dsDNA
TFII transcription factor for RNA Polymerase
II
TATA DNA sequence found in the core promoter
region 25bp upstream of transcription start
point

• Formation of PIC
• Complex is ready to transcribe, certain factors
  dissociate, forming what the authors call the
  scaffold which is part of reinitiation process
• Initiation and reinitiation involve same
  transcription machinery but proceed in different
  processes

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Mediator Stimulates CTD Phosphorylation

Mediator is a combination of SRB (Suppressor of
RNAPolymerase B), Med (mediator protein), and
other proteins it allows transcription to be
responsive to activators and stimulates RNA Pol
II carboxy-terminal domain (CTD) phosphorylation.
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Phosphorylation patterns orchestrate TFs
CTD (carboxy-terminal domain)
RNA Pol II
Hemali P. Phatnani et al. Genes Dev. 2006 20
2922-2936

• CTD is an extension of the C terminus of RNAPII
• Flexible binding platform for factors binding
  determined by the phosphorylation patterns CTD
• Changes in phosphorylation patterns while
  transcription occurs is believed to orchestrate
  the association of different sets of
  transcription factors (http//www.genesdev.org/cgi
  /content/full/20/21/2922)

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Authors questions?
For efficient gene transcription, RNA Pol II
depends on high rates of transcription initiation
and reinitiation reinitiation is proposed to
follow a different pathway, including an
intermediate that acts as a platform/scaffold.
1. Are there transcription factors bound to the
template after initiation?
2. If so, are they part of a reinitiation
process?
3. How do initiation complexes dissociate into
reinitiation intermediate/scaffolds?
4. How do the activators affect the reinitiation
intermediate?
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Fig. 1 Experiment Preinitiation Complex
?Scaffold
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PIC formation immobilized promoter template
assay

• Procedure
• Immobilize template
• Incubate with Gal4-AH activator and NE (nuclear
  extract) for 40 min
• Wash (to remove any unbound components)
• Add NTPs, incubate 2 min
• Wash again
• Digest with PstI, 30 min, 37oC
• ? Western Blot (gel electrophoresis, transfer to
  membrane)
• Only one round of transcription occurred 1 min
  incubation of PICs, NTPs, and sarkosyl to block
  reinitiation step produced same transcription
  signal

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PIC dissociates into a reinitiation intermediate
? scaffold
Figure 1
Lane 1 negative control (no template)
Lane 2 positive control (template present,
bands represent PIC formation

• Lane 3 template nucleotides
• RNA Pol II, TFIIB, TFIIF dissociated from
  templates (red box)
• TFIID, TFIIA, Gal4-AH remained (blue box), TFIIE,
  TFIIH are weakly bound (lighter bands)

PIC
SCAFFOLD
Lane 4 template ATP (same effect as NTPs)

• Lane 5 template AMPPNP
• This ATP Analog did not promote PIC dissociation
  (resembles lane 2), suggests that ATP
  hydrolysis, rather than transcription, is
  necessary for PIC dissociation

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Structure of AMPPNP
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Are components of scaffold required for
reinitiation?
? PIC ? Scaffold formed addition of NTPs
Addition of 2nd Nuclear Extract separate strains
containing mutatation in the highlighted factors
(i.e. absence of mediator, TFIID, etc)
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Does the Scaffold function in Reinitiation?
Figure 1 Experiment
Figure 2 Experiment
Addition of 2nd NE Mutated (normal) ?Srb2
(mediator) Srb4ts(mediator)G41E(TFIIB)
Kin28ts(TFIIH) Tfa1ts(TFIIE) I143N(TBP)
Toal-25(TFIIA)
Figure 2
Assayed by primer extension, 5 end of primer
labled, autoradiograph
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Figure 2 Experiment Continued
2nd NE ?Srb2 (mediator), Srb4ts(mediator),
G41E(TFIIB), Kin28ts(TFIIH), Tfa1ts(TFIIE),
I143N(TBP), Toal-25(TFIIA)
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Figure 2 Experiment Take home message

• Mutated strains were created to analyze the
  transcription factors that they believed where
  part of the scaffold
• Transcription appears weak (or not at all) in 1st
  NE because mutant extracts are unable to form
  PICs (data not shown)
• If transcription appears stronger in the 2nd NE,
  that is evidence that the missing TF is present
  in the scaffold
• Exception 1 TFA1st (TFIIE) appeared weaker in
  2nd NE ? but this data and figure 1 data shows
  that TFIIE is weakly bound
• Exception 2 G41E (TFIIB), weak transcription
  signal observed TFIIB is not required in the
  scaffold but is sufficient for transcription

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How do Initiation complexes dissociate into
scaffolds?
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How do Initiation complexes dissociate into
scaffolds?
Fig. 1 lane 4 5 ATP hydrolysis rather than
transcription is required for PIC dissociation.
Fig. 3
Identified 3 subunits of TFIIH that may be good
candidates for ATP-dependent activity Rad25,
Rad3 and Kin28
PIC intermediates that lacked both Kin28 Tfb1
subunits of TFIIH formed in nuclear extracts that
contained the mutant subunits
PICs lacking TFIIH were not able to dissociate
into scaffolds
PIC dissociation is dependent on ATP and TFIIH
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Summary of events for Fig. 4 Experiment

• Figure 4A
• Scaffolds obtained in the same manner as in fig.
  1 experiment
• - incubate immobilized templates with GAL4-AH,
  GAL4-VP16, or no activator
• Scaffolds incubated in transcription buffer
  (containing all NTPs, TFs) for indicated times
• Wash for 1 min
• Western blot
• Figure 4B
• WT Nuclear extract is incubated with template and
  different activators or no activator
• NTPs added, samples removed for primer extension
  at different times
• TS (y-axis) plotted versus time (minutes)

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Activators affect Reinitiation
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Biphasic kinetics of transcription
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Conclusions

• Scaffold/reinitiation intermediate consists of
  TFIID, TFIIA, Mediator, TFIIH, TFIIE (weakly)
  whereas, TFIIB, TFIIF dissociate

• TFIIE is present in the scaffold/reinitiation
  complexas the least stable component

• ATP hydrolysis, rather than transcription, is
  necessary for PIC dissociation

• PIC dissociation is dependent on ATP and TFIIH
  (though unable to determine which subunit of
  TFIIH is responsible)

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Conclusions

• Activator plays dual role in promoting high
  levels of reinitiation can promote recruitment
  of transcription machinery, and can stabilize
  scaffold complex to promote reinitiation

• Rates of reinitiation are higher than initiation

• Presence of scaffold stimulates initial rates of
  transcription 2-3 fold

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Future Directions

• Determine which subunit of TFIIH is responsible
  for PIC dissociation

• Confirm function of holopolymerase in the
  initiation/reinitiation step

• Confirm if reinitiation involves the recruitment
  of free RNA Pol II, or RNA Pol II in a distinct
  complex, to the scaffold

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Questions?