Transcript Lecture PowerPoint to accompany Molecular Biology Fifth Edition Robert F. Weaver Chapter 11 General Transcription Factors in Eukaryotes Copyright © The McGraw-Hill Companies, Inc.

Lecture PowerPoint to accompany
Molecular Biology
Fifth Edition
Robert F. Weaver
Chapter 11
General Transcription
Factors in Eukaryotes
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Transcription in Eukaryotes
• Eukaryotic RNA polymerases, unlike their
bacterial counterparts, are incapable of
binding by themselves to their respective
promoters
• Eukaryotic RNA polymerases rely on
proteins called transcription factors to
show them the way
• Two classes: general transcription factors
and gene-specific transcription factors
(activators)
11-2
11.1 Class II Factors
• General transcription factors combine with
RNA polymerase to form a preinitiation
complex
– This complex is able to initiate transcription
when nucleotides are available
– Tight binding involves formation of an open
promoter complex with DNA at the
transcription start site that has melted
• The assembly of preinitiation complexes
involving polymerase II is quite complex
11-3
The Class II Preinitiation Complex
• Class II preinitiation complex contains:
– RNA Polymerase II
– 6 general transcription factors:
• TFIIA, TFIIB, TFIID, TFIIE, and TFIIH
• The transcription factors (TF) and
polymerase bind the preinitiation complex
in a specific order (as studied in vitro)
11-4
Four Distinct Preinitiation Complexes
• Transcription factors bind to class II promoters in
the following order in vitro:
• TFIID with help from TFIIA binds to the TATA box
forming the DA complex
• TFIIB binds next generating the DAB complex
• TFIIF helps RNA polymerase bind to a region
from -34 to +17, now it is DABPolF complex
• Last the TFIIE then TFIIH bind to form the
complete preinitiation complex = DABPolFEH
• In vitro, the participation of TFIIA seems to be
optional
11-5
Model of Formation of the DABPolF
Complex
11-6
Structure and Function of TFIID
TFIID contains several subunits
– TATA-box binding protein (TBP)
• Highly evolutionarily conserved
• Binds to the minor groove of the TATA box
– Saddle-shaped TBP lines up with DNA
– Underside of the saddle forces open the minor
groove
– The TATA box is bent into 80° curve
– TBP-associated factors (TAFs) specific for
class II
11-7
Structure of the TBP-TATA box complex
11-8
The Versatility of TBP
• Genetic studies have demonstrated TBP
mutant cell extracts are deficient in:
– Transcription of class II genes
– Transcription of class I and III genes
• TBP is a universal transcription factor
required by all three classes of genes
• Required in transcription of at least some
genes of Archaea, single-celled organisms
lacking nuclei
11-9
The TBP-Associated Factors
• These are also called TAFs (TAFIIs is written to
denote transcription of class II genes)
• 13 TAFs have been identified and associated
with class II preinitiation complexes
• The core TAFs were first named according to
their molecular mass but have now been
renamed according to their sizes, from largest to
smallest
• Several functions discovered:
– Interaction with the core promoter elements
– Interaction with gene-specific transcription factors
– When attached to TBP extend the binding of TFIID
beyond the TATA box
11-10
Model for the Interaction Between TBP
and Promoters
11-11
Roles of TAF1 and TAF2
• The TAF1 and TAF2 help the TFIID bind to
the initiator and DPE of promoters
• They enable TBP to bind to TATA-less
promoters that contain elements such as a
GC box
• Different combinations of TAFs are
required to respond to variosu activators,
at least in higher eukaryotes
• TAF1 has two enzymatic activities:
– Histone acetyltransferase (HAT)
– Protein kinase
11-12
Transcription Enhancement by Activators
11-13
Exceptions to the Universality of TAFs
and TBP
• TAFs are not universally required for
transcription of class II genes
• Even TBP is not universally required
• Some promoters in higher eukaryotes respond
to an alternative protein such as TRF1 (TBPrelated factor 1)
• The general transcription factor NC2:
– Stimulates transcription from DPE-containing
promoters
– Represses transcription from TATA-containing
promoters
11-14
Structure and Function of TFIIB
• Structural studies have revealed that TFIIB
binds to TBP at the TATA box via its Cterminal domain and polymerase II via its
N-terminal domain
• The protein provides a bridging action that
effects a coarse positioning of polymerase
active center about 25 –30 bp downstream
of the TATA box
• Plays an important role in establishing the
transcription start site
11-15
TFIIB Domains
• A loop motif of the N-terminal domain in
TFIIB effects a fine positioning of the
transcription start by interacting with
template ssDNA near the active center
• TFIIB N-terminal domain, finger and linker
domains, lies close to the RNA
polymerase II active center and to largest
subunit of TFIIF in preinitiation complex
11-16
TFIIH
• TFIIH is the last general transcription factor to
join the preinitiation complex (contains 9
subunits)
• Separates into 2 complexes
• Protein kinase complex of 4 subunits
• Core TFIIH complex of 5 subunits with 2 DNA
helicase/ATPase activities
• Plays two major roles in transcription initiation:
– Phosphorylates the CTD of RNA polymerase II
– Unwinds DNA at the transcription start site to create
the “transcription bubble”
11-17
Phosphorylation of the CTD of RNA
Polymerase II
• The preinitiation complex forms with
hypophosphorylated form of RNA
polymerase II (IIA)
• Then TFIIH phosphorylates serines 2 and
5 in the heptad repeat in the carboxylterminal domain (CTD) of the largest RNA
polymerase subunit
– This creates the phosphorylated form of the
polymerase enzyme (IIO)
– This phosphorylation is essential for initiation
of transcription
11-18
Phosphorylated Polymerase IIO During
Elongation
• During the shift from initiation to elongation,
two serines of the CTD are phosphorylated
(serines 2 and 5 - and sometimes serine 7)
• Evidence exists that transcription
complexes near the promoter have CTDs in
which serine 5 is phosphorylated but that
this phosphorylation shifts to serine 2 as
transcription progresses
• TFIIH phosphorylates serine 5 and CTDK-1
(in yeast) phosphorylates serine 2
11-19
Role of TFIIE and TFIIH
TFIIE and TFIIH are not essential for the
formation of an open promoter complex or
for elongation
• Required for promoter clearance
• TFIIH has 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
11-20
Participation of General Transcription
Factors in Initiation
• TFIID with TFIIB, TFIIF and RNA
polymerase II form a minimal initiation
complex at the initiator
• Addition of TFIIH, TFIIE and ATP allow
DNA melting at the initiator region and
partial phosphorylation of the CTD of
largest RNA polymerase subunit
• These events allow production of abortive
transcripts as the transcription stalls at
about +10
11-21
Expansion of the Transcription Bubble
• Energy is provided by ATP
• DNA helicase of TFIIH causes unwinding
of the DNA
• Expansion of the transcription bubble
releases the stalled polymerase
• Polymerase is now able to clear the
promoter
11-22
Transcription Factors in Elongation
• Elongation complex continues elongating
the RNA when:
– Polymerase CTD is further phosphorylated by
TEFb
– NTPs are continuously available
• TBP and TFIIB remain at the promoter
• TFIIE and TFIIH are not needed for
elongation and dissociate from the
elongation complex
11-23
Model for the participation of GTFs in initiation,
promoter clearance, and elongation
11-24
The Mediator Complex and the RNA
Polymerase II Holoenzyme
• Mediator is a collection of proteins also
considered to be a general transcription
factor as it is a part of most class II
preinitiation complexes
• Mediator is not required for initiation, but it
is required for activated transcription
• It is possible to assemble the preinitiation
complex adding general transcription
factors to RNA polymerase II holoenzyme
11-25
Eukaryotic Control of Transcription
• Eukaryotes control transcription primarily at the
initiation step
• There is also some control exerted during
elongation, which can involve overcoming
transcription pausing or transcription arrest
• RNA polymerases do not transcribe at a steady
rate as they pause, sometimes for a long time,
before resuming transcription
• Tend to pause at pause sites or DNA sequences
that destabilize the DNA-RNA hybrid and cause
the polymerase to backtrack
11-26
Promoter Proximal Pausing
• A sizable fraction of genes contain specific pause
sites lying 20-50bp downstream of the
transcription start site
• Two protein factors are known to help stabilize
RNA polymerase II in the paused state - DRB
sensitivity inducing factor (DSIF) and negative
elongation factor (NELF)
• The signal to leave the paused state is delivered
by the positive elongation factor-b (P-TEFb), which
is a protein kinase that can phosphorylate
polymerase II, DSIF, and NELF
11-27
TFIIS Stimulates Proofreading of
Transcripts
• TFIIS stimulates proofreading, the
correction of misincorporated nucleotides,
likely by stimulating RNase activity of the
RNA polymerase
• This would allow polymerase to cleave off
a misincorporated nucleotide and replace
it with a correct one
11-28
11.2 Class I Factors
• The preinitiation complex that forms at
rRNA promoters is much simpler than the
preinitiation complex for class II RNA
polymerase
• It involves polymerase 1 plus two
additional transcription factors:
– A core-binding factor, SL1 or TIF-IB
– A UPE-binding factor, upstream-binding factor
(UBF) or upstream activating factor (UAF)
11-29
The Core-Binding Factor
• The core-binding factor, SL1, was
originally isolated on the basis of its ability
to direct polymerase initiation
• SL1 also shows species specificity
• This factor is the fundamental
transcription factor required to recruit
RNA polymerase I
11-30
Upstream-Binding Factor (UBF)
• This transcription factor is an assembly
factor that helps the core binding factor to
bind to the core promoter element
• It works by bending the DNA dramatically
• Degree of reliance on UBF varies
considerably from one organism to another
• Human UBF is a transcription factor that
stimulates transcription by polymerase I
and can activate the intact promoter, or the
core element alone, and it mediates the
activation by the UPE
11-31
Structure and Function of SL1
• Human SL1 is composed of TBP and three
TAFs (TAFI110, TAFI63, TAFI48) which
bind TBP tightly
• These TAFs are completely different from
those found in TFIID
• Yeast and other organisms have TAFIs
that are different from the human group
11-32
11.3 Class III Factors
• In 1980 a transcription factor was found
that bound to the internal promoter of the
5S rRNA gene and stimulated its
transcription – TFIIIA
• Two other transcription factors TFIIIB and
TFIIIC have also been studied
• Transcription of all classical class III genes
requires TFIIIB and TFIIIC
• Transcription of 5S rRNA genes requires all
three
11-33
TFIIIA
• TFIIIA was the first eukaryotic transcription
factor to be discovered
• First member of the family of DNA-binding
proteins that feature a zinc finger to be
described
– Zinc finger is roughly finger-shaped protein
domain that contains 4 amino acids that bind
a zinc ion
– Has nine zinc fingers that appear to insert into
the major groove on either side of the
promoter for the 5S rRNA gene
11-34
TFIIIB and TFIIIC
• Both of these transcription factors are
required for transcription of the classical
polymerase III genes
• They depend on each other for their
activities
• TFIIIC is an assembly factor that allows
TFIIIB to bind to the region just upstream
of the transcription start site
• TFIIIB can remain bound and sponsor
initiation of repeated transcription rounds
11-35
Scheme for Assembly of the Preinitiation
Complex on a classical class II promoter
• TFIIIC binds to
internal promoter
• TFIIIC promotes
binding of TFIIIB with
its TBP
• TFIIIB promotes
polymerase III binding
at start site
• Transcription begins
11-36
Model of Preinitiation Complex on
TATA-Less Promoter
• Assembly factor binds first
• Another factor, containing
TBP, is now attracted
• Complex now sufficient to
recruit polymerase except
for class II
• Transcription begins
11-37
The Role of TBP
• Assembly of the preinitiation complex on each
kind of eukaryotic promoter begins with binding
of an assembly factor to the promoter
• TBP is this factor with TATA-containing class II
and class III promoters
• If TBP is not the first bound, it still becomes part
of the growing preinitiation complex and serves
an organizing function
• Specificity of TBP depends on associated TAFs
11-38