molecular biology fourth edition chapter 11 general transcription factors in eukaryotes lecture...
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Molecular BiologyFourth Edition
Chapter 11
General Transcription Factors in Eukaryotes
Lecture PowerPoint to accompany
Robert F. Weaver
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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 transcription start site melted
• While the class II complex is quite involved, explore it first, then those of classes I and III
11-3
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
11-4
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
11-5
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
– 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
• Required in transcription of at least some genes of the Archaea, single-celled organisms lacking nuclei
11-8
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
11-9
Model for the Interaction Between TBP and Promoters
11-10
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
11-11
Transcription Enhancement by Activators
11-12
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 (TBP-related factor 1)
• The general transcription factor NC2: – Stimulates transcription from DPE-containing
promoters
– Represses transcription from TATA-containing promoters
11-13
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
11-14
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
11-15
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
11-16
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 carboxyl-terminal 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
11-17
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
11-18
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
11-19
Role of TFIIE and TFIIH
TFIIE and TFIIH
• Not essential for – Formation of an open promoter complex – Elongation
• Required for 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
Schematic Model
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
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
11-26
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
11-27
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
11-28
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)
11-29
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
• 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
11-31
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
11-32
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 feature to be described– Zinc finger is roughly finger-shaped protein
domain – Contains 4 amino acids that bind a zinc ion
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 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
11-36
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 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
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