General transcription factors in Eukaryotes

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Chapter 11

• General transcription factors in Eukaryotes
• Zhang xiaorong

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• Eukaryotic RNA polymerases ,unlike their
  prokaryotic counterparts ,are incapable of
  binding by themselves to their respective
  promoters .instead they rely on proteins called
  transcription factors to show them the way .Such
  factors are grouped into two classes
• general transcription factors
• gene-specific transcription factors

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11.1 classII factors

• a. The classII preinititation complex
• b. Structure and function of TFIID
• c. Structure and function of TFIIA and TFIIB
• d. Structure and function of TFIIF
• e. Structure and function of TFIIE and TFIIH
• f. Elongation factors
• g. The polymerase II holoenzyme

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a.The classII preinititation complex

• The classII preinitiation complex contains
  polymerase II and 6 general transcription factors
  named
• TF?A,TF?B,TF?D,TF?E,TF?F,TF?H.

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a. The DABPolFcomplex
b.The DBPolFEH
Building the
preinitiation complex
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Transcription factors bind to class II
promoters, including the adenovirus major late
promoter ,in the following order in vitro

• (1) TFIID,with help of TFIIA,binds to the TATA
  box ,forming the DA complex.
• (2) TFIIB binds next ,causing minimal
  perturbation of the protein-DNA interaction.
• (3) TFIIF helps RNA polymerase bind to a region
  extending from at least position-34 to
  position17.
• (4) TFIIE and TFIIH , forming the DABPolFEH
  preinitiation complex. The participation of TFIIA
  seems to be optional in vitro.

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Footprinting the DA and DAB complexes
Caution TFIIB perturb the DNA structure to
alter its susceptibility to DNase attack.(10)
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• Footprinting the DABPolF complex
• when RNA polymerase and
• TFIIF joined the complex ,they caused a large
  extension of the footprint ,to about position 17
  

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• Model for formation of the DABPolF complex

Polymerase II(red) and TFIIF(green)bind
cooperatively,by forming a binary complex that
joins the preformed DAB complex.
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Structure and function of TFIID

• TFIID is a complex protein containing TBP and
  8-10 TBP-associated factors (TAFIIs).
• The TATA-Box-Binding protein is highly
  evolutionarily conserved
• TBP from yeast ,fruit flies ,and human have more
  than 80 identical in amino acid sequence
• Yeast TBP functions well in a preinitiation
  complex in which all the other transcription
  factors are mammalian

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TBP

• How does the TBP in TFIID bind to the TATA box?
  
• TBP contacts with the base pairs in the major
  groove or minor groove

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TBP bind to the minor groove of the TATA box,and
not the major groove of the TATA box.
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How does TFIID associate with the TATA box minor
groove?
The saddle-shaped TBP lines up with the DNA, and
the underside of the saddle forces open the minor
groove and bends the TATA box into an 80-degree
curve.
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• Just like a saddle sit on a horse

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The versatility of TBP

• TBP is a universal transcription factor required
  by all three classes of genes.
• TBP mutant cell extracts are deficient,not
  only in transcription of classII genes,but also
  in transcription of classI and classIII genes
• a. Locations of the mutations
• b-e. Effects of the mutations.

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The TBP associated factors

• TFIID contains at least eight TAFIIs,in addition
  to TBP.Most of these TAFIIs are evolutionarily
  conseved in the eukaryotes.

Relationships among the TAFs of fruit
filies,humans,and yeast.
Structure of Drosophlia TFIID
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• TFIID worked much better than TBP in supporting
  transcription from the promoter contains Inr and
  DPE
• Because TFIID contains both TBP and TAF

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The function of TAFIIs

• TAFII250 and TAFII150 help TFIID bind to the
  initiator and downstream elements of promoters
  and therefore can enable TBP to bind to TATA-less
  promoters that contain such elements.
• TAFII250 and TAFII110 help TFIID interact with
  sp1 that is bound to GC boxes upstream of the
  transcription start site.These TAFIIs therefore
  ensure that TBP can bind to TATA-less promoters
  that have GC boxes.
• TAFII250 has two enzymatic activities. It is a
  histone acetyltransferase and a protein kinase.

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• TAFII250 and TAFII150 bound to the Hsp70 promoter
  and became labeled
• TBP alone cant be labeled
• indicate that TAFIIs are responsible for
  recognizing the initiator and DPE

Radioactively labeled DNA contain Hsp70 promoter
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• TBP caused a footprint only in the TATA Box
• The ternary complex caused an additional
  footprint in the initiator and downstream element

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• The TBP part of TFIID is of course important in
  recognizing the majority of the well-studied
  classII promoter
• What about promoters that lack a TATA Box?
• TATA less promoters contain other elements
  that ensure the binding of TBP (initiators and
  DPEs or upstream element )

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(c)
(a)
(b)
a.TATA-containing promoter.
b.TATA-less promoter with initiator element.
c.TATA-less promoter with GC boxes.
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• TBP-TAFII250-TAFII150 complex functioned almost
  as well as the TFIID in recognizing a promoter
  composed of a TATA box and an initiator

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??

A model for transcription enhancement by
activator
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Exceptions to the universality of TAFs and TBP

• Exceptions
• TAFs do not appear to be universally
  required for transcription of classII genes.
• TBP is not universally required ,some
  promoters in higher eukaryotes respond to an
  alternative protein such as TRF1 and not to TBP.
• Some promoters can be stimulated by a
  TBP-free TAFII-containing complex (TFTC),rather
  than by TFIID.

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• The central role of TBP in forming
  preinitiation complex has been further challenged
  by the discovery of a TBP-free TAFII-containing
  complex(TFTC) that is able to sponsor
  preinitiation complex without any help of TFIID
  or TBP

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Three dimensional models of TFIID and TFTC.
TFIID(green)
TFTC(blue)
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c. Structure and function of TFIIA and TFIIB

• TFIIA contains two subunits(yeast),or three
  subunits(fruit flies and humans).This factor may
  be more properly considered a TAFII because it
  binds to TBP and stabilizes binding between TFIID
  and promoters.
• TFIIB serves as a linker between TFIID and
  TFIID/polymeraseII .It has two domains,one of
  which is responsible for binding to TFIID,the
  other for continuing the assembly of the
  preinitiation complex.

Hypothetical structure of a TFIIA-TFIIB-TBP-TATA
box complex.
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This structure shows TFIIA and TFIIB binding to
the upstream and downstream stirrups,respectively,
of TBP.This puts two factors in advantageous
positions to perform their functions.
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d. Structure and function of TFIIF

• TFIIF is actually composed of two
  polypeptides,called RAP30 and RAP70.
• It can reduce nonspecific interactions
  between polymerase and DNA.
• TFIID can interact with both polymerase? and
  the DAB complex so it can lead polymeraseII to
  promoters

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e.Structure and function of TFIIE and TFIIH

• Structure
• TFIIE, composed of two molecules each of a 34-kD
  and a 56-Kd polypeptide, binds after polymerase
  and TFIIF. Both subunits are required for binding
  and transcription stimulation.
• TFIIH is the last general transcription factor to
  join the preinitiation complex.
• The preinitiation complex envisioned by Tjian and
  Reinberg.

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(No Transcript)
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Function

• TFIIH has a DNA helicase activity that is
  essential for transcription,presumably because it
  causes full melting of the DNA at the promoter
  and thereby facilitates promoter clearance.
• A subunit of TFIIH phosphorylates the
  carboxyl-terminal domain (CTD)of the largest RNA
  polymeraseII subunit.TFIIE greately stimulates
  this process in vitro.

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• TFIIH alone is sufficient to cause some
  polymerase phosphorylation ,but the other
  factors enhances phosphorylation.
• TFIIE fiercily stimulate phosphorylation

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• a increasing amount of polymerase
• PolB has no CTD and cant be phosphorylated
• Pol II O has many phosphorylated aa
• b ?????? cleaved and we know that
  phosphorylation is take place in CTD

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• TFIIH is a complex protein ,both structurally and
  functionally ,it can be separated into two
  complex
• protein kinase complex
• Core complex with DNA helicase / ATPase
  activity

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• 1 heat-denature , 2 no protein
• 3 only RAD25 4 10 ng RAD25 and ATP
• 5 20 ng RAD25 and ATP (RAD25 is a subunit of
  TFIIH )

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f. Elongation factors
1.TFIIS stimulates elongation

• TFIIS ,stimulates elongation by limiting long
  pauses at discrete sites.TFIIF also stimulates
  elongation,apparently by limiting transient
  pausing.

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• Effect of IIS on transcription initiation and
  elongation combined.

Effect of IIS on transcription elongation.
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2.TFIIS stimulates proofreading of transcripts

• TFIIS stimulates proofreading-the correction of
  misincorporated nucleotides-presumably by
  stimulating the RNase activity of the RNA
  polymerase, allowing it to cleave off a
  misincorporated nucleotide (and perhaps other
  nucleotides)and replace it with the correct one.

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• the polymerase alone was able to do some
  proofreading
• TFIIs stimulates proofreading of the transcript

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A model for proofreading by RNA polymerase II
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g. The polymerase II holoenzyme

• Yeast and mammalian cells have an RNA polymerase
  II holoenzyme that contains many polypeptides in
  addition to the subunits of the polymerase .The
  yeast holoenzyme contains a subunit of general
  transcription factors and at least some of the
  SRB proteins.The rat holoenzyme contains all the
  general transcription factors except TFIIA.

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• Purified yeast RNA polymerase II
• 1 core polymerase
• 2 holoenzyme

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A model for the participation of general
transcription factors in initiation,
promoter,clearance,and elongation
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THE END

• Thanks!!!