Molecular Biology Fourth Edition Chapter 11 General Transcription Factors in Eukaryotes Lecture...
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Transcript of Molecular Biology Fourth Edition Chapter 11 General Transcription Factors in Eukaryotes Lecture...
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