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Chapt 11 General Transcription Factors in Eukaryotes

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Chapt 11 General Transcription Factors in Eukaryotes Student learning outcomes: Explain how General Transcription Factors (GTFs): Attract RNAP to promoters – PowerPoint PPT presentation

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Title: Chapt 11 General Transcription Factors in Eukaryotes


1
Chapt 11 General Transcription Factors in
Eukaryotes
  • Student learning outcomes
  • Explain how General Transcription Factors (GTFs)
  • Attract RNAP to promoters
  • Dictate direction and starting point of
    transcription
  • Responsible for basal level of transcription
  • (gene-specific activators regulate level of
    transcription)
  • Explain that GTFs for Pol II (mRNA) include
  • TFs IIA, IIB, IID, IIE, IIF, IIH TBP, TAFIIs,
    mediator, IIS

2
  • Explain that GTFs for Pol I (rRNA, snRNA)
    include
  • SL1 (TIF-1B) and UBF
  • Explain that GTFs for Pol III (tRNA, 5S rRNA)
    include
  • TFIII A, B and C
  • Describe briefly new techniques (from Chapt. 5)
  • DNase footprinting, EMSA (mobility shift assay),
    S1 nuclease, primer extension, Run-off
    transcription
  • Impt. Figures 1, 2, 4, 7, 8, 9, 11, 12, 13,
    14, 25, 26, 28, 29, 32, 38 39, 42
  • Review Q 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 14, 15,
    16, 18, 21, 22, 24, 26, 28, 29, 30, 33, 35, 38
    AQ 1, 2

3
  • EMSA electrophoretic mobility shift assay
  • can identify specific protein complexes
    binding DNA
  • complexes formed in vitro are analyzed on 4
    PAG
  • (nondenaturing gels)
  • antibodies to specific proteins assist
    analysis

Shift
Fig. 5.36
4
DNase I footprinting - where proteins bind
  • DNA labeled at one end
  • add increasing amounts of protein
  • cleave with DNase I (nonspecific cleavage),
  • compare with DNA seq ladder

Fig. 5.37
5
S1 nuclease - 5 end of specific transcript
S1 is nonspecific Dnase for ss DNA
  • Probe longer than expected transcript
  • labeled at one end
  • Hybridize to transcript
  • Add S1
  • Resolve on gel

Fig. 5.27
6
Primer extension assay identifies start of
specific transcript, relative amounts
  • Primer specific to transcript
  • (made in vitro or in vivo)
  • Reverse transcribe
  • See size of product on gel

Fig. 5.30
7
Run-off transcription Identifies start of
transcription in vitro, relative amounts
Fig. 5.31
8
11.1 Class II Factors (pol II makes mRNA)
  • General transcription factors (GTFs) combine with
    pol II to form preinitiation complex (PIC)
  • Initiates transcription when NTPs available
  • Tight binding -gt formation of RPo (open promoter
    complex), melted DNA at transcription start site
  • Class II preinitiation complex contains
  • Pol II
  • 6 general transcription factors (each
    multisubunit)
  • TFIIA TFIIB TFIID TFIIE TFIIF TFIIH
  • (Named for biochemical fractionation peaks
  • needed for basal transcription by pol II)

9
Four ordered Distinct Preinitiation
Complexes identified from in vitro expts with
pure proteins
  • Model promoter AdML (adenovirus major late,
    which has TATA, Inr and DPE)
  • TFIID TFIIA binds to TATA box forms DA complex
  • TFIIB binds next -gt DAB complex
  • TFIIF helps pol II bind -34 to 17 DABPolF
    complex
  • Last, TFIIE then TFIIH bind to form complete
    preinitiation complex DABPolFEH
  • In vitro, TFIIA seems to be optional

10
EMSA assays identify components of PIC AdML model
promoter pure proteins
Fig. 1
11
DNase footprinting identifies region bound DAB
binds about -20 to -35 DABpolF to 17
Figs. 2, 3
12
Model of Formation of DABPolF Complex
Fig. 4
13
Structure and Function of TFIID complex TBP
TAFIIs
  • TATA-box binding protein (TBP) Highly
    evolutionarily conserved
  • Binds minor groove of TATA box
  • Saddle-shaped TBP on DNA
  • Underside of saddle forces open
  • minor groove
  • TATA box is bent into 80 curve
  • 8 to 10 TBP-associated factors (TAFIIs) specific
    for class II

Fig. 6 TBPTATA TBP green TATA orange other
DNA blue
14
Versatility of TBP (38-kD)
  • Genetic studies demonstrated TBP mutant cell
    extracts (ts mutants) are deficient in
  • Transcription of class II genes (even if no TATA)
  • Transcription of class I and III genes (no TATA)
  • TBP is 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 Archaea also have IIB-like protein
    evolutionarily closer to eukaryotes that to
    Bacteria

15
TBP-Associated Factors (TAF11S) for pol II
  • 8 proteins named by MW
  • Most evolutionarily conserved in eukaryotes
  • Identified by immuno-ppt TBP
  • Several functions
  • Interact with core promoter
  • Interact with gene-specific transcription factors
  • When attached to TBP, extend binding of TFIID
    beyond TATA box (footprint)

Fig. 8
16
Conservation of TAFIIS
TFIID (TBP TAFs) stimulates transcription off
Inr DPE promoters
Fig. 9
Fig. 10
17
TAFs stimulate binding of TBP to promoters
  • TAFII250 and TAFII150 help TBP bind to initiator
    and DPE of promoters
  • TAFII250 has enzymatic activities
  • Histone acetyltransferase
  • Protein kinase (itself, TFIIF)
  • TAFII110 aids TFIID interaction with Sp1 bound to
    GC boxes upstream of transcription start
  • TAFs enable TBP to bind to
  • TATA-less promoters that contain elements such as
    GC box

Fig. 12 Hsp70 promoter
18
Model for Interaction of TBP and Promoters TAFs
aid or recruit TBP
Fig. 13
19
TFIID can respond to many activators of
transcription (Chapt. 12) Different TAFs are
required for different Activators
Fig. 14 NTF-1 uses TAFII 150 or TAF11 60 SP1
uses TAFII110 Other activators use other TAFs
20
Exceptions to Universality of TAFIIs and TBP
  • TAFs not universally required for 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 in Drosophila nerual tissue)
  • TFTC (TBP-free TAFII-containing complex) can
    promote PIC
  • General transcription factor NC2
  • Stimulates transcription from DPE-containing
    promoters
  • Represses transcription from TATA-containing
    promoters

21
Different requirements for TAFs for yeast
expression (ts TAFs mutants tested) Rpb1 is
required for all class II transcription
22
Structure and Function of TFIIB
  • TFIIB (35 kD) is a single polypeptide
  • TFIIB binds to
  • TBP at TATA box via its C-terminal domain
  • Pol II via its N-terminal domain (finger)
  • Single strand DNA template
  • TFIIB positions pol II active center 25 30 bp
    downstream of TATA box

23
TFIIB Domains
C-terminal domain binds to TBP at TATA box
N-terminal domain binds to pol II And ss DNA
Fig. 17 pol II regions include clamp, dock,
wall, and the grey shaded TBP green
24
TFIIH is a complex factor
  • TFIIH last GTF to join preinitiation complex
    (PIC)
  • 2 major roles in transcription initiation
  • Phosphorylates CTD of pol II (IIa -gt IIo)
  • Unwinds DNA at transcription start site to create
    transcription bubble
  • Contains 9 subunits in 2 complexes
  • Protein kinase complex of 4 subunits
  • Core TFIIH complex of 5 subunits has 2 DNA
    helicase/ATPase activities

25
TFIIH Phosphorylates CTD of Pol II
Fig. 22 Pol IIb lacks CTD
  • PIC forms with hypo-phosphorylated pol II (IIA)
  • TFIIH phosphorylates serines 2 and 5 in the
    heptad repeat in CTD of Rpb1, largest RNAP
    subunit
  • creates phosphorylated form of pol II (IIo)
  • phosphorylation essential for initiation

26
Phosphorylated Pol IIO During Elongation
  • TFIIH phosphorylates ser2 and 5 to initiate
  • During shift from initiation to elongation,
    phosphorylation on serine 5 of heptad repeat is
    lost, removed by a phosphatase
  • If phosphorylation of serine 2 is also lost, pol
    II pauses until rephosphorylation by a non-TFIIH
    kinase occurs (pTEFb)

27
Initiation
  • TFIID with TFIIB, TFIIF and pol II form minimal
    initiation complex at initiator
  • Addition of TFIIH, TFIIE and ATP allow DNA
    melting initiator region, phosphorylation of pol
    II CTD (Rpb1)
  • Allow production of abortive transcripts
    transcription stalls at about 10

Fig. 25 part
28
Expansion of Transcription Bubble
  • Energy from ATP
  • DNA helicase of TFIIH causes unwinding of DNA
  • Expanded transcription bubble releases stalled
    pol II
  • Pol II can now clear promoter

Fig. 25 part
29
Model of Initiation, Promoter Clearance,
Elongation
  • Elongation needs NTPs, pTEFb to phosphorylate
  • TBP and TFIIB at promoter
  • TFIIE and TFIIH dissociate

Fig. 25
30
Model Assembly of GTFs and pol II at promoter
transcription from R to L
Fig. 26 Roger Kornberg
31
Mediator Complex and Pol II Holoenzyme Roger
Kornberg lab Rick Young lab
  • Mediator
  • Collection of 20 proteins considered GTF
  • Found often as part of class II preinitiation
    complexes
  • not required for initiation,
  • is required for activated transcription (Chapt.
    12)
  • Possible to assemble preformed preinitiation
    complex by adding some GTFs to pol II holoenzyme
  • then add with TBP, TFIIB, E and H to promoter

32
Elongation Factor TFIIS
  • Eukaryotes control transcription primarily at
    initiation
  • Some control at elongation
  • TFIIS, isolated from tumor cells, specifically
    stimulates transcription elongation
  • TFIIS stimulates proofreading of transcripts,
    likely by stimulating RNase activity of pol II
  • Proofreading correction of misincorporated
    nucleotides, by cleaving off a few and replacing
    3

33
Elongation and TFIIS
  • Pol II not transcribe steady rate
  • Short stops in transcription transcription
    pauses
  • Pauses for variable lengths of time
  • Tend to occur at defined pause sites where DNA
    sequence destabilizes RNA-DNA hybrid, causing pol
    II to backtrack
  • If backtracks too far, pol II cannot recover
    alone Transcription arrest
  • Pol II needs help from TFIIS during transcription
    arrest

Fig. 28
This marks the end of Pol II section
34
11.2 Class I Factors
  • RNA polymerase I plus 2 transcription factors
    make up preinitiation complex
  • much simpler than PIC for pol II
  • Pol I has many subunits, some shared with pol II
    and pol III (Table 10.2)
  • Transcription factors
  • A core-binding factor, SL1 (humans) or TIF-IB
  • A UPE-binding factor,
  • upstream-binding factor (UBF in mammals)
  • or upstream activating factor (UAF in yeast)

35
Core-Binding Factor SL1 (Bob Tjian)
  • Originally isolated by ability to direct pol I
    initiation
  • Species specificity
  • Fundamental transcription factor required to
    recruit pol I to promoter

Fig. 31 in vitro transcription promoters
contain small insertions, deletions primer
extension assay C and T DNA seq a no promoter
36
Upstream-Binding Factor (UBF) is assembly factor
  • UBF helps SL1 bind to core promoter element
  • Bends DNA dramatically
  • Degree of reliance on UBF varies among organisms
  • 97-kD polypeptide

Fig. 32 footprint rRNA gene enhanced DNase
cleave
37
Structure and Function of SL1
  • Human SL1 TBP and TAFs which bind TBP tightly
  • TAFI110
  • TAFI63
  • TAFI48
  • TAFIs different from those in TFIID
  • Yeast, other organisms have different TAFIs

Fig. 35 immuno-ppt SL1 with anti-TBP antibody
dissociate and re-ppt (6, 7)
38
11.3 Class III Transcription Factors (TFIII s)
  • TFIIIA transcription factor bound to internal
    promoter of 5S rRNA gene, stimulated its
    transcription in vitro (Bob Roeder)
  • Two other transcription factors TFIIIB and TFIIIC
  • Transcription of tRNA genes requires only TFIIIB
    and TFIIIC
  • Transcription of 5S rRNA genes requires all three

Fig. 10.26
39
TFIIIA
  • First eukaryotic transcription factor discovered
  • First member of family of DNA-binding proteins
    that feature zinc motif (Chapt. 12)
  • Zinc finger is finger-shaped protein domain
  • Contains 4 amino acids that bind zinc ion
  • TFIIIA has 2 Cys, 2 His (others have 4 Cys)
  • Finger binds major groove of DNA

40
TFIIIB and TFIIIC
  • Both are required for transcription of classical
    pol III genes
  • Depend on each other for activity
  • TFIIIC is assembly factor that allows TFIIIB to
    bind just upstream of transcription start site
  • TFIIIB can remain bound, help initiate repeated
    transcription rounds

Fig. 38 footprint on tRNA genes (yellow) lane d
has heparin added to remove loose proteins
41
Assembly of Preinitiation Complex (PICIII)
  • TFIIIC (huge protein) binds to internal promoter
    (boxes A and B)
  • TFIIIC promotes binding of TFIIIB with its TBP
  • TFIIIB promotes pol III binding at start site
  • Transcription begins

Fig. 39
42
Preinitiation Complexes can form on TATA-Less
Promoter
  • Assembly factor binds
  • Another factor, containing TBP, is now attracted
  • Complex is sufficient to recruit polymerase
    (except for some class II genes)
  • Transcription begins

43
The Role of TBP
  • Assembly of preinitiation complex (PIC )on each
    type of eukaryotic promoter begins with binding
    of assembly factor(s)
  • TBP is this factor with TATA-containing class II
    and class III promoters
  • If TBP is not first bound protein, it still
    becomes part of growing PIC and serves organizing
    function
  • Specificity of TBP depends on associated TAFs

44
Conclusion eukaryotic transcription is really
complex compared to prokaryotes
  • General Transcription Factors (GTF or TF)
  • Attract different RNA polymerases to promoters
  • Dictate direction and starting point of
    transcription
  • Responsible for basal level of transcription
  • (gene-specific activators control level of
    transcription)
  • GTF vary for promoters/ pol of 3 classes
  • Pol II IIA, IIB, IID, IIE, IIF, IIH TBP,
    TAFIIs, mediator, IIS
  • Pol I SL1 (has TBP) and UBF
  • Pol III TFIII A, IIIB (has TBP) and IIIC

45
Review questions
  • 1. Describe in order the proteins that assemble
    in vitro to form class II preinitiation complex.
  • Describe role of TBP and the TAFIIs
  • Describe DNase footprint, S1 nuclease experiment.
  • Compare class I and class III factors
  • 26. What is the holoenzyme pol II, and how does
    it differ from the core pol II?
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