General Transcription Factors in Eukaryotes

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Chapter 11

• General Transcription Factors in Eukaryotes

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Class I factors
(UPE)
SL1
Upstream-binding factor (UBF)
SL1
Direct transcription from the rRNA promoter in
combination with Pol I SL1 does not bind
promoter DNA by itself SL1 is specie-specific
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Human SL1 does not work on mouse rRNA promoter
Mouse extract
Mouse rRNA promoter
Human rRNA promoter
Core
UCE
In vitro transcription
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(Upstream-binding factor) UBF
UBF is composed of 97kD and 94 kD proteins 97kD
protein is sufficient for binding to Core element
and Upstream control element (UCE) UBF
stimulates transcription of the rRNA gene in the
presence or absence of UCE.
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What is SL1? How does TBP fit in?
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Structure and Function of SL1
SL1 is composed of TBP and three TAFs (TAFI110,
TAFI63, and TAFI48)
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Immunodepletion of SL1 prevent rRNA
transcription TBP alone cannot restore SL1
function
Antibody against TBP
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How do you find out what else are in SL1?

1. Gel shift analysis
2. DNase I footprinting analysis
3. In vitro transcription assay
4. BrdU photo cross-linking assay
5. Co-immunoprecipitation assay

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Three TAFIs (TBP associated factors) are
important for the SL1 activity
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TBP uses completely different sets of TAFs for
RNA pol I and RNA pol II Two sets of TAFs
compete for binding to the TBP
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Class III Factors
Three factors, TFIIIA, TFIIIB, and TFIIIC, are
important for transcription of 5s rRNA gene.
TFIIIA is not required for transcription of
tRNA genes TFIIIB and TFIIIC are important for
all Pol III genes
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TFIIIA is important for 5S rRNA synthesis but not
tRNA synthesis
Nonspecific Ab
Nonspecific Ab
TFIIIA Ab
TFIIIA Ab
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In vitro transcription
Frog Oocyte extract
Somatic cell extract
TFIIIA is a zinc finger DNA binding protein
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What are the roles of TFIIIB and TFIIIC?
TFIIIC helps TFIIIB bind to the promoter After
that, TFIIIB by itself binds to the promoter
tightly without TFIIIC
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heparin
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TFIIIC can bind internal elements (Box A and Box
B) TFIIIB can not bind DNA by itself but can do
so in the presence of TFIIIC (binds to the
upstream region right near the transcription
start) Once it binds to DNA, TFIIIB can stay
bound in the absence of TFIIIC Promoter bound
TFIIIB can initiate multiple rounds of
transcription The order of binding of TFIII
factors to the Pol III gene is important for the
transcription. TFIIIB is composed of TBP and
TAFs (TAF-172 and TAF-L)
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TFIIIC is a large protein complex with six
subunits It has a dumbbell shape with two
globular domains connected by a stretchable
linker.
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Three unifying principle for preinitiation
complex formation

• It always starts with an assembly factor that
  recognizes a specific binding site in the
  promoter
• TATA-containing class II promoters TBP
• TATA-less class II promoters TAFs or upstream
  element binding factors such as Sp1
• Class I promoter UBF
• Class III promterTFIIIC (TFIIIA)
• 2) TBP is always involved in the assembly
  process, playing an organizing role in
  preinitiation complex formation.
• 3) The specificity of TBP is governed by its
  associated factors (TAFs)

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