Transcript Inquiry into Life Twelfth Edition

Lecture PowerPoint to accompany
Molecular Biology
Fourth Edition
Robert F. Weaver
Chapter 11
General Transcription
Factors in Eukaryotes
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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 transcription
start site melted
• While the class II complex is quite
involved, explore it first, then those of
classes I and III
11-2
The Class II Preinitiation
Complex
• Class II preinitiation complex contains:
– Polymerase II
– 6 general transcription factors:
•
•
•
•
•
TFIIA
TFIIB
TFIID
TFIIE
TFIIH
• The transcription factors (TF) and
polymerase bind the preinitiation complex
in a specific order
11-3
Four Distinct Preinitiation
Complexes
• 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
11-4
Model of Formation of the
DABPolF Complex
11-5
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
– 8 to 10 copies of TBP-associated factors
(TAFIIs) specific for class II
11-6
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 the Archaea, single-celled
organisms lacking nuclei
11-7
The TBP-Associated Factors
• These are also called TAFIIs
• 8 different proteins are designated by MW
• Most are evolutionarily conserved in
eukaryotes
• 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
11-8
Model for the Interaction
Between TBP and Promoters
11-9
Roles of TAFII250 and TAFII150
• The TAFII250 and TAFII150 help the TFIID bind
to the initiator and DPE of promoters
• Also aid in TFIID interaction with Sp1 that is
bound to GC boxes upstream of the transcription
start site
• They enable TBP to bind to:
– TATA-less promoters that contain elements such as a
GC box
• TAFII250 has 2 enzymatic activities:
– Histone acetyltransferase
– Protein kinase
11-10
Transcription Enhancement by
Activators
11-11
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
11-12
Structure and Function of TFIIB
• The gene for human TFIIB has been
cloned and expressed by Reinberg et al.
• TFIIB binds to
– TBP at the TATA box via its C-terminal domain
– 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
11-13
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
11-14
TFIIH
• TFIIH is the last general transcription
factor to join the preinitiation complex
• Plays 2 major roles in transcription
initiation:
– Phosphorylate the CTD of RNA polymerase II
– Unwind DNA at the transcription start site to
create the transcription bubble
11-15
Phosphorylation of the CTD of
RNA Polymerase II
• The preinitiation complex forms with
hypophosphorylated form of RNA
polymerase II
• 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
11-16
of transcription
Phosphorylated Polymerase IIO
During Elongation
• During the shift from initiation to
elongation, phosphorylation on serine 5 of
the heptad repeat is lost
• If phosphorylation of serine 2 is also lost,
polymerase pauses until
rephosphorylation by a non-TFIIH kinase
occurs
11-17
TFIIH
TFIIH is a very complex protein
– 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
11-18
Role of TFIIE and TFIIH
TFIIE and TFIIH
• Not essential for
– Formation of an open promoter complex
– Elongation
• Required for promoter clearance
11-19
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
11-20
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
11-21
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
11-22
Schematic Model
11-23
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
11-24
The Elongation Factor TFIIS
• Eukaryotes control transcription primarily
at the initiation step
• There is some control exerted at
elongation
• TFIIS, isolated from tumor cells,
specifically stimulates transcription
11-25
Elongation and TFIIS
• RNA polymerases do not transcribe at steady rate
• Short stops in transcription are termed
transcription pauses
– Pauses are for variable lengths of time
– Tend to occur at defined pause sites where DNA
sequence at those sites destabilize the RNA-DNA
hybrid, causing polymerase to backtrack
• If backtracking goes too far, polymerase cannot
recover on its own = Transcription arrest
• Polymerase needs help from TFIIS during a
transcription arrest
11-26
TFIIS Stimulates Proofreading
of Transcripts
• TFIIS stimulates proofreading, 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
• Proofreading is the correction of
misincorporated nucleotides
11-27
11.2 Class I Factors
• RNA polymerase I and 2 transcription
factors make up the preinitiation complex,
much simpler than the preinitiation
complex for class II RNA polymerase
• Transcription factors:
– A core-binding factor, SL1 or TIF-IB
– A UPE-binding factor, upstream-binding factor
(UBF) or upstream activating factor (UAF)
11-28
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
11-29
Upstream-Binding Factor
• This transcription factor is an assembly
factor that helps SL1 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
• Size of polypeptide is 97-kD
11-30
Structure and Function of SL1
• Human SL1 is composed of TBP and
TAFs which bind TBP tightly:
– TAFI110
– TAFI63
– TAFI48
• These TAFs are completely different from
those found in TFIID
• Yeast and other organisms have TAFIs
that are different from the human group
11-31
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
11-32
TFIIIA
• TFIIIA was the first eukaryotic transcription
factor to be discovered
• First member of the family of DNA-binding
proteins that feature a zinc feature to be
described
– Zinc finger is roughly finger-shaped protein
domain
– Contains 4 amino acids that bind a zinc ion
11-33
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
11-34
Scheme for Assembly of
Preinitiation Complex
• TFIIIC binds to
internal promoter
• TFIIIC promotes
binding of TFIIIB with
its TFB
• TFIIIB promotes
polymerase III binding
at start site
• Transcription begins
11-35
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
11-36
The Role of TBP
• Assembly of the preinitiation complex on each
kind of eukaryotic promoter begins with binding
of assembly factor to 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
11-37