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

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Presentation on theme: "Chapt 11 General Transcription Factors in Eukaryotes Student learning outcomes: Explain how General Transcription Factors (GTFs): Attract RNAP to promoters."— Presentation transcript:

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, TAF II s, mediator, IIS 11-1

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 11-2

3 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

4 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

5 Fig. 5.27 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

6 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

7 Fig. 5.31 Run-off transcription Identifies start of transcription in vitro, relative amounts

8 11-8 **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 -> 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 11-9 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 -> 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 11-10 Fig. 1

11 DNase footprinting identifies region bound DAB binds about -20 to -35; DABpolF to +17 11-11 Figs. 2, 3

12 11-12 Model of Formation of DABPolF Complex Fig. 4

13 11-13 Structure and Function of TFIID complex = TBP + TAF II s 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 (TAF II s) specific for class II Fig. 6 TBP:TATA TBP green; TATA orange; other DNA blue

14 11-14 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 11-15 TBP-Associated Factors ( TAF 11 S ) 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 TAF II S 11-16 Fig. 10 Fig. 9 TFIID (TBP + TAFs) stimulates transcription off Inr DPE promoters

17 11-17 TAFs stimulate binding of TBP to promoters TAF II 250 and TAF II 150 help TBP bind to initiator and DPE of promoters TAF II 250 has enzymatic activities: –Histone acetyltransferase –Protein kinase (itself, TFIIF) TAF II 110 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 11-18 Model for Interaction of TBP and Promoters; TAFs aid or recruit TBP Fig. 13

19 11-19 TFIID can respond to many activators of transcription (Chapt. 12) Different TAFs are required for different Activators Fig. 14; NTF-1 uses TAF II 150 or TAF11 60; SP1 uses TAF II 110; Other activators use other TAFs

20 11-20 Exceptions to Universality of TAF II s 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 TAF II -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 11-21

22 11-22 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 11-23 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 II And ss DNA

24 11-24 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 -> 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 11-25 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

26 11-26 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 11-27 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 11-28 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

29 11-29 Model of Initiation, Promoter Clearance, Elongation Fig. 25 Elongation needs NTPs, pTEFb to phosphorylate TBP and TFIIB at promoter TFIIE and TFIIH dissociate

30 Model: Assembly of GTFs and pol II at promoter; transcription from R to L 11-30 Fig. 26; Roger Kornberg

31 11-31 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 11-32 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 11-33 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. 28This marks the end of Pol II section

34 11-34 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 11-35 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 11-36 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 11-37 Structure and Function of SL1 Human SL1 = TBP and TAFs which bind TBP tightly: –TAF I 110 –TAF I 63 –TAF I 48 TAF I s different from those in TFIID Yeast, other organisms have different TAF I s Fig. 35; immuno-ppt SL1 with anti-TBP antibody; dissociate and re-ppt (6, 7)

38 11-38 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 11-39 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 11-40 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 11-41 Assembly of Preinitiation Complex (PIC III ) 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 11-42 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 11-43 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, TAF II s, mediator, IIS Pol I: SL1 (has TBP) and UBF Pol III: TFIII A, IIIB (has TBP) and IIIC 11-44

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 TAF II s 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? 11-45

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