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 -> 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 -> 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 TBP:TATA
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
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 -> 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
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
This marks the end of Pol II section
Fig. 28

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

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?