Chapt 11General 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

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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

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Fig. 5.36

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. 5.37

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. 5.27

S1 nuclease - 5’ end of specific transcriptS1 is nonspecific Dnase for ss DNA

• Probe longer than expected transcript; • labeled at one end• Hybridize to transcript;• Add S1;• Resolve on gel

Fig. 5.30

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. 5.31

Run-off transcriptionIdentifies start of transcription in vitro,relative amounts

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**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 -> 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)

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Four ordered Distinct Preinitiation Complexesidentified 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 -> 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

EMSA assays identify components of PICAdML model promoter; pure proteins

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Fig. 1

DNase footprinting identifies region boundDAB binds about -20 to -35; DABpolF to +17

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Model of Formation of DABPolF Complex

Fig. 4

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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 TBP:TATA

TBP green; TATA orange; other DNA blue

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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

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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

Conservation of TAFIIS

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Fig. 9

TFIID (TBP + TAFs) stimulates transcription off Inr DPE promoters

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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

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Model for Interaction of TBP and Promoters; TAFs aid or recruit TBP

Fig. 13

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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

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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

Different requirements for TAFs for yeast expression; (ts TAFs mutants tested)Rpb1 is required for all class II transcription

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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

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TFIIB Domains

Fig. 17: pol II regions include clamp, dock, wall, and the grey shaded; TBP green

C-terminal domain binds to TBP at TATA box; N-terminal domain binds to pol IIAnd ss DNA

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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 -> 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

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TFIIH Phosphorylates CTD of Pol II

• 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

Fig. 22; Pol IIb lacks CTD

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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)

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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

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Expansion of Transcription Bubble

Fig. 25 part

• Energy from ATP• DNA helicase of TFIIH causes unwinding of DNA• Expanded transcription bubble releases stalled pol II• Pol II can now clear promoter

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Model of Initiation, Promoter Clearance, Elongation

Fig. 25

• Elongation needs NTPs, pTEFb to phosphorylate• TBP and TFIIB at promoter• TFIIE and TFIIH dissociate

Model: Assembly of GTFs and pol II at promoter; transcription from R to L

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Fig. 26; Roger Kornberg

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Mediator Complex and Pol II HoloenzymeRoger 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

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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’

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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. 28This marks the end of Pol II section

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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)

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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 assayC and T = DNA seq; a = no promoter

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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

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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)

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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

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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

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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

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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

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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

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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

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

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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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