Transcript Inquiry into Life Twelfth Edition

Molecular Biology
Lecture 17
Chapter 11
General Transcription
Factors in Eukaryotes
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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
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
Model of Formation of the
DABPolF Complex
11-4
11-5
The polymerase extends
the footprint to +17
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
– 8 to 10 copies of TBP-associated factors
(TAFIIs) specific for class II
11-7
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
11-8
TBP-TATA box complex
Overall bend of 80 degrees on the DNA caused by TBP11-9
TBP
In-vitro
transcription assays
11-10
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
11-11
Structure of TFIID
11-12
Most TAF proteins
Are conserved from
Yeast to human
11-13
Adenovirus promoters
In-vitro
transcription assays
Requirement of TAFs for efficient transcription on AdML
11-14
and Hsp70 promoters
Roles of TAFII250 and TAFII150
• The TAFII250 and TAFII150 help 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
11-15
DNAse 1 footprinting
on the hsp70 promoter
11-16
bhTBP = TBP produced in bacteria
vhTBP = TBP produced in virus infected cells
11-17
Activation by SP1 requires TAF 110
11-18
Model for the Interaction
Between TBP and Promoters
11-19
Transcription Enhancement by
Activators
11-20
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
11-21
Function of TFIIB – Binds to TBP and recruits polymerase
Function of TFIIA – Stabilizes TBP-TATA box binding
11-22
Function of TFIIF – Made of two protein RAP30 and 70
RAP 30 sigma-like binds to E. coli polymerase required for
Binding of RAP70 to core enzyme.
TFIIF reduces non-specific interaction of polymerase with DNA
11-23
TFIIH
• TFIIH is the last general transcription
factor to join the preinitiation complex with
TTIIE
• 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
11-24
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
11-25
of transcription
Function of TFIIH (9 subunits)
And TFIIE
11-26
Function of TFIIH – Phosphorylation of CTD of PolII
11-27
The TFIIH helicase activity is required for transcription in yeast
Permissive
temperature
Nonpermissive
temperature
11-28
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
11-29
TFIIS stimulates elongation
11-30
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
11-31
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
11-32
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
11-33
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
11-34
Schematic Model
11-35
Class I Factors
• RNA polymerase I and III 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)
11-36
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
11-37
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
11-38
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
11-39
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
11-40
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
11-41
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
11-42
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
11-43