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

2. 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 transcription start site melted
While the class II complex is quite involved, explore it first, then those of classes I and III

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

4. Four Distinct Preinitiation Complexes
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

5. Model of Formation of the DABPolF Complex

6. 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
8 to 10 copies of TBP-associated factors (TAFIIs) specific for class II

7. 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 the Archaea, single-celled organisms lacking nuclei

8. The TBP-Associated Factors
These are also called TAFIIs
8 different proteins are designated by MW
Most are evolutionarily conserved in eukaryotes
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

9. Model for the Interaction Between TBP and Promoters

10. Roles of TAFII250 and TAFII150
The TAFII250 and TAFII150 help the TFIID bind to the initiator and DPE of promoters
Also aid in TFIID interaction with Sp1 that is bound to GC boxes upstream of the transcription start site
They enable TBP to bind to:
TATA-less promoters that contain elements such as a GC box
TAFII250 has 2 enzymatic activities:
Histone acetyltransferase
Protein kinase

11. Transcription Enhancement by Activators

12. 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

13. Structure and Function of TFIIB
The gene for human TFIIB has been cloned and expressed by Reinberg et al.
TFIIB binds to
TBP at the TATA box via its C-terminal domain
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

14. 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

15. TFIIH
TFIIH is the last general transcription factor to join the preinitiation complex
Plays 2 major roles in transcription initiation:
Phosphorylate the CTD of RNA polymerase II
Unwind DNA at the transcription start site to create the transcription bubble

16. Phosphorylation of the CTD of RNA Polymerase II
The preinitiation complex forms with hypophosphorylated form of RNA polymerase II
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

17. Phosphorylated Polymerase IIO During Elongation
During the shift from initiation to elongation, phosphorylation on serine 5 of the heptad repeat is lost
If phosphorylation of serine 2 is also lost, polymerase pauses until rephosphorylation by a non-TFIIH kinase occurs

18. TFIIH TFIIH is a very complex protein 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

19. Role of TFIIE and TFIIH TFIIE and TFIIH Not essential for
Formation of an open promoter complex
Elongation
Required for promoter clearance

20. 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

21. 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

22. 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

23. Schematic Model

24. 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

25. The Elongation Factor TFIIS
Eukaryotes control transcription primarily at the initiation step
There is some control exerted at elongation
TFIIS, isolated from tumor cells, specifically stimulates transcription

26. Elongation and TFIIS RNA polymerases do not transcribe at steady rate
Short stops in transcription are termed transcription pauses
Pauses are for variable lengths of time
Tend to occur at defined pause sites where DNA sequence at those sites destabilize the RNA-DNA hybrid, causing polymerase to backtrack
If backtracking goes too far, polymerase cannot recover on its own = Transcription arrest
Polymerase needs help from TFIIS during a transcription arrest

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

28. 11.2 Class I Factors
RNA polymerase I and 2 transcription factors make up the preinitiation complex, much simpler than the preinitiation complex for class II RNA polymerase
Transcription factors:
A core-binding factor, SL1 or TIF-IB
A UPE-binding factor, upstream-binding factor (UBF) or upstream activating factor (UAF)

29. 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

30. Upstream-Binding Factor
This transcription factor is an assembly factor that helps SL1 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
Size of polypeptide is 97-kD

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

32. 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

33. TFIIIA
TFIIIA was the first eukaryotic transcription factor to be discovered
First member of the family of DNA-binding proteins that feature a zinc feature to be described
Zinc finger is roughly finger-shaped protein domain
Contains 4 amino acids that bind a zinc ion

34. 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

35. Scheme for Assembly of Preinitiation Complex
TFIIIC binds to internal promoter
TFIIIC promotes binding of TFIIIB with its TFB
TFIIIB promotes polymerase III binding at start site
Transcription begins

36. 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

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