Eukaryotic Transcription General Factors and Mechanism

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Eukaryotic TranscriptionGeneral Factors and
Mechanism
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Eukaryotes Have Three RNA Polymerases
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Basal Transcription in Eukaryotes
A. Structure of RNA polymerase II

• B. Transcription Initiation
• The six general transcription factors for RNA
  pol II
• TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH
• Plus the Mediator Complex

C. Promoter clearance, elongation, and termination
D. Class I and class III factors
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RNA Polymerase Subunits
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Structure of RNA Polymerase II
Kornberg and colleagues, 2001
12 subunits in intact RNAP II Rpb4 7 are
absent from structure Names are (RNA Polymerase B)
Rpb1, 2, and 3 are homologous to ?, ?, and ?
of bacterial RNAP
Rpb1 includes unique, extended C-terminal
domain containing 26-52 repeats of
YSPTSPS (important later)
Rpb5, 6, 8, 10, and 12 are found in all three
eukaryotic polymerases
Fig. 10.10
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The Basic Machinery of RNA Polymerase IISix
General Transcription Factors
Modified from Callebaut et al. BMC Genomics 2005
6100
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Basal Promoter Structure in Eukaryotes
Not all promoters have all elements
Mammals have well-defined start site, but yeast
do not
Smale and Kadonaga, Ann. Rev. Biochem (2003),
72449-79
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Step-by-step Formation of Preinitiation Complex
By Class II Factors (1991)
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Model for Assembly of Preinitiation Complex
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Footprinting to Define Binding Sites (1990)
Footprinting by reaction with hydroxyl radicals
(from 1,10-phenanthroline-Cu2) or with DNaseI
Protection appears on both strands from TFIIA
and TATA-box binding protein (TBP) from TFIID
TFIIB gives little further change, but one
nucleotide (10) becomes exposed to DNase I
Experiments used yeast TBP expressed in E. coli
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RNA Polymerase Gives Extensive Protection From
DNase I
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Model for Assembly of Preinitiation Complex
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Structure and Function of TFIID
TATA-box-binding protein (TBP)
80 identity of TATA-box-binding domain from
yeast to human
Up to 14 TBP-associated factors (TAFIIs)
Subscript designation necessary because TBP
functions in transcription of class I and III
genes
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Structure of the Complex Between TBP and TATA DNA
Initial proposed structure of complex based on
TBP structure (1992)
Structure of co-crystal (1993)
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TAFIIs A Central Component of TFIID
IP to TBP revealed 8 TAFIIs in Drosophila
Gel shows recombinant proteins used to assemble
TFIID
TAFIIs have two main functions 1. DNA
binding 2. Binding other activators They also
can have enzymatic activities, like HAT activity
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DNA Binding by TAFIIs
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DNA Binding by TAFIIs
Photocrosslinking using 5-BrdU
X-links to TAFII250 and TAFII150
When TAFIIs were omitted, no crosslinks were
observed
TBP doesnt crosslink, presumably because it
binds in the minor groove
Footprinting showed that TBP binds, and TAFIIs
gave expanded footprint
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Diversity of Promoter Binding
A. With TATA
B. w/o TATA
C. With activator
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Mediator Complex an Additional General
Transcription Factor and Co-activator
First hint came from squelching, inhibition
of one activator by high concentration of another
(1988)
Isolated as complex of 20 subunits, gt 1000 kDa
(1994)
Cryo-EM structure with RNAP II (2002)
Calls into question direct interactions of
activators with TAF components of TFIID
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Exceptions to the Universality of TAFs and TBP
Mutations in some TAFs decrease transcription
of surprisingly few genes.
TBP is not found in all preinitiation
complexes. Drosophila encodes an alternative TBP
(TRF1, TBP-related factor 1).
There has also been found a TBP-free
TAFII-containing complex, which can promote
transcription without TBP.
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TFIIA Enhances Binding by TBP
Reported to be dispensable in vitro
Essential in yeast
Relieves autoinhibition of TBP
Relatively simple only two subunits
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TFIIB Structure and Function
Just a single polypeptide of 35 kDa
Binds after TFIID and TFIIA
Essential for RNA polymerase II binding
Two domains N-terminal domain binds RNA
polymerase C-terminal domain contacts TBP and
bent DNA
TFIIBN
TBP
TFIIBC
Bushnell et al (Kornberg), 2004
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TFIIF Associates Tightly with RNA Polymerase
Cryo-EM (16 Å) of RNAPII-TFIIF (2003)
TFIIF in blue, linkers between domains not
resolved at this resolution
Conformational changes in RNAP upon TFIIF
binding shown in yellow
One subunit of TFIIF (Tgf2 in yeast, Rap30 in
human) may be a homolog of bacterial ? factor
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Model for Assembly of Preinitiation Complex
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TFIIH Structure and Function
Last general transcription factor to bind in
vitro
Plays two major roles Phosphorylates the
C-terminal domain of RNA polymerase Unwinds DNA
at initiation site to create transcription
bubble
TFIIE Helps TFIIH bind
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Phosphorylation of the CTD of RNA Polymerase II
Phosphorylation is stimulated by other factors,
notably TFIIE
Phosphorylation is even observed of a fusion
protein of the CTD with GAL4 transcription factor
Two serine residues in repeats are
phosphorylated (S2 and S5) S5 is heavily
phosphorylated early in elongation and S2 is
phosphorylated later
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TFIIH Creation of the Transcription Bubble
Of nine subunits, four are kinase- and five are
helicase-associated
One helicase subunit, RAD25 in yeast, is
essential and has helicase activity
No protein
No ATP
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Model for General Transcription
FactorsInitiation, Promoter Clearance, and
Elongation
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Retention of a Scaffold Complex Allows
Efficient Re-initiation
Subsequent initiation events can be more
efficient because most of initiation complex is
already formed
Hahn, Nat. Struct. Mol. Biol (2004) 11, 394-403
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One More Factor Elongation Factor TFIIS
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One More Factor Elongation Factor TFIIS
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Models For Termination of Transcription on mRNAs
Model 1 polyA signal leads to changes in
composition of RNAP
Model 2 mRNA is cleaved at polyA signal,
generating new 5-end that is rapidly degraded
(torpedo model)
Buratowski, Curr. Opin. Cell Biology (2005) 17,
257-261.
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Class I factors There Are Only Two
Core binding factor SL1 in human
Isolated in 1985 as one of two fractions needed
for transcription in vitro from a human rRNA
template
Composed of three TAFs (TAFI110, TAFI63, and
TAFI48) and TBP
Upstream binding factor UBF in human
Probably only one polypeptide 97 kDa
Binds to upstream promoter element (UPE) to
enhance initiation
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Class III Factors Three Identified
TFIIIA
First eukaryotic transcription factor identified
First DNA-binding protein found to contain zinc
fingers (has 9)
Binds specifically to internal promoters of 5S
rRNA genes
TFIIIB and TFIIIC
Both required for transcription and depend on
each other
TFIIIB contains TBP
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Common Themes in Eukaryotic Transcription
Initiation
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Key Points
1. Initiation of eukaryotic transcription
involves the formation of large complexes at the
promoter. These complexes generate tremendous
versatility.
2. Of the three transcription classes, class II
is the most complicated but also the best
understood. At least six factors are required,
most of which are complexes of several
polypeptides.
3. A common theme is that TBP plays an important
role. It can be involved in initiation either by
binding to the TATA sequence of by associating
with other factors.
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