1. Molecular Biology Fifth Edition
Lecture PowerPoint to accompany
Molecular Biology Fifth Edition
Robert F. Weaver
Chapter 11
General Transcription Factors in Eukaryotes
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

2. Transcription in Eukaryotes
Eukaryotic RNA polymerases, unlike their bacterial counterparts, are incapable of binding by themselves to their respective promoters
Eukaryotic RNA polymerases rely on proteins called transcription factors to show them the way
Two classes: general transcription factors and gene-specific transcription factors (activators)

3. 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 the transcription start site that has melted
The assembly of preinitiation complexes involving polymerase II is quite complex

4. The Class II Preinitiation Complex
Class II preinitiation complex contains:
RNA Polymerase II
6 general transcription factors:
TFIIA, TFIIB, TFIID, TFIIE, and TFIIH
The transcription factors (TF) and polymerase bind the preinitiation complex in a specific order (as studied in vitro)

5. Four Distinct Preinitiation Complexes
Transcription factors bind to class II promoters in the following order in vitro:
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

6. Model of Formation of the DABPolF Complex

7. 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
TBP-associated factors (TAFs) specific for class II

8. Structure of the TBP-TATA box complex

9. 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 Archaea, single-celled organisms lacking nuclei

10. The TBP-Associated Factors
These are also called TAFs (TAFIIs is written to denote transcription of class II genes)
13 TAFs have been identified and associated with class II preinitiation complexes
The core TAFs were first named according to their molecular mass but have now been renamed according to their sizes, from largest to smallest
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. Model for the Interaction Between TBP and Promoters

12. Roles of TAF1 and TAF2
The TAF1 and TAF2 help the TFIID bind to the initiator and DPE of promoters
They enable TBP to bind to TATA-less promoters that contain elements such as a GC box
Different combinations of TAFs are required to respond to variosu activators, at least in higher eukaryotes
TAF1 has two enzymatic activities:
Histone acetyltransferase (HAT)
Protein kinase

13. Transcription Enhancement by Activators

14. 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

15. Structure and Function of TFIIB
Structural studies have revealed that TFIIB binds to TBP at the TATA box via its C-terminal domain and 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
Plays an important role in establishing the transcription start site

16. 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

17. TFIIH
TFIIH is the last general transcription factor to join the preinitiation complex (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
Plays two major roles in transcription initiation:
Phosphorylates the CTD of RNA polymerase II
Unwinds DNA at the transcription start site to create the “transcription bubble”

18. Phosphorylation of the CTD of RNA Polymerase II
The preinitiation complex forms with hypophosphorylated form of RNA polymerase II (IIA)
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

19. Phosphorylated Polymerase IIO During Elongation
During the shift from initiation to elongation, two serines of the CTD are phosphorylated (serines 2 and 5 - and sometimes serine 7)
Evidence exists that transcription complexes near the promoter have CTDs in which serine 5 is phosphorylated but that this phosphorylation shifts to serine 2 as transcription progresses
TFIIH phosphorylates serine 5 and CTDK-1 (in yeast) phosphorylates serine 2

20. Role of TFIIE and TFIIH
TFIIE and TFIIH are not essential for the formation of an open promoter complex or for elongation
Required for promoter clearance
TFIIH has DNA helicase activity that is essential for transcription, presumably because it causes full melting of the DNA at the promoter and thereby facilitates promoter clearance

21. 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

22. 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

23. 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

24. Model for the participation of GTFs in initiation, promoter clearance, and elongation

25. 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

26. Eukaryotic Control of Transcription
Eukaryotes control transcription primarily at the initiation step
There is also some control exerted during elongation, which can involve overcoming transcription pausing or transcription arrest
RNA polymerases do not transcribe at a steady rate as they pause, sometimes for a long time, before resuming transcription
Tend to pause at pause sites or DNA sequences that destabilize the DNA-RNA hybrid and cause the polymerase to backtrack

27. Promoter Proximal Pausing
A sizable fraction of genes contain specific pause sites lying 20-50bp downstream of the transcription start site
Two protein factors are known to help stabilize RNA polymerase II in the paused state - DRB sensitivity inducing factor (DSIF) and negative elongation factor (NELF)
The signal to leave the paused state is delivered by the positive elongation factor-b (P-TEFb), which is a protein kinase that can phosphorylate polymerase II, DSIF, and NELF

28. TFIIS Stimulates Proofreading of Transcripts
TFIIS stimulates proofreading, the correction of misincorporated nucleotides, 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

29. 11.2 Class I Factors
The preinitiation complex that forms at rRNA promoters is much simpler than the preinitiation complex for class II RNA polymerase
It involves polymerase 1 plus two additional transcription factors:
A core-binding factor, SL1 or TIF-IB
A UPE-binding factor, upstream-binding factor (UBF) or upstream activating factor (UAF)

30. 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

31. Upstream-Binding Factor (UBF)
This transcription factor is an assembly factor that helps the core binding factor 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
Human UBF is a transcription factor that stimulates transcription by polymerase I and can activate the intact promoter, or the core element alone, and it mediates the activation by the UPE

32. Structure and Function of SL1
Human SL1 is composed of TBP and three TAFs (TAFI110, TAFI63, TAFI48) which bind TBP tightly
These TAFs are completely different from those found in TFIID
Yeast and other organisms have TAFIs that are different from the human group

33. 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

34. TFIIIA
TFIIIA was the first eukaryotic transcription factor to be discovered
First member of the family of DNA-binding proteins that feature a zinc finger to be described
Zinc finger is roughly finger-shaped protein domain that contains 4 amino acids that bind a zinc ion
Has nine zinc fingers that appear to insert into the major groove on either side of the promoter for the 5S rRNA gene

35. 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

36. Scheme for Assembly of the Preinitiation Complex on a classical class II promoter
TFIIIC binds to internal promoter
TFIIIC promotes binding of TFIIIB with its TBP
TFIIIB promotes polymerase III binding at start site
Transcription begins

37. 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

38. The Role of TBP
Assembly of the preinitiation complex on each kind of eukaryotic promoter begins with binding of an assembly factor to the 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