1. General Transcription Factors in Eukaryotes
Chapter 11

2. Class II factors
General transcription factors combine with RNA polymerase to form a preinitiation complex
Initiates transcription when nucleotides are available
Tight binding involves formation of an open promoter complex with DNA at transcription start site melted to allow polymerase to read it
This complex is able to initiate transcription when nucleotides are available

3. The Class II Preinitiation Complex
Class II preinitiation complex contains: specific order
Polymerase II
6 general transcription factors:
TFIIA
TFIIB
TFIID
TFIIE
TFIIF
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 to +17 = DABPolF complex
Lastly - TFIIE and TFIIH bind to form the complete preinitiation complex = DABPolFEH
In vitro the participation of TFIIA seems to be optional. Danny Reinberg and Philip Sharp performed DNA gel mobility shift and DNase and hydroxyl radical footprinting experiments

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
8 to 10 copies of TBP-associated factors (TAFIIs) specific for class II
Conserved in yeast, fruit flies, plants and humans have more than 80% identical amino acid sequence.

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
To identify TAFIIs from Drosophila cells, Tjian and his collegues used an antibody specific for TBP to immunoprecipitate TFIID from a crude TIID preparation. Then they treated the immunoprecipitate with 2.5 M urea to strip TAFs off the TBP-antibody precipitate and displayed TAFs by SDS-PAGE. This had led to identification of eight TAFs.

9. Homework
Explain model for the interaction Between TBP and promoters – page 290
Explain transcription enhancement by activators – page 291
Hint : Roles of TAFII250 and TAFII150
Explain Figure – page 301

10. Model for the Interaction Between TBP and Promoters
How does TFIID interact with various parts of promoter or promoter elements?

11. Roles of TAFII250 and TAFII150 in TATA less promoters
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
How does TFII D bind to TATA box? In TATA containing promoter there are elements that ensure binding of TAFII250 and 150 to TATA and Inr. In TATA less promoters, the factors bind to initiator and DPE because TBP cannot by itself bind to Inr. In case of TATA-less promoter with GC boxes, TBP cannot bind to this promoter by itself. The whole TFIID can bind to this promoter through interactions with Sp1 bound at GC boxes.

12. Transcription Enhancement by Activators
NTF-1 binds to TAFII250 and 150 or 250 and 60 to activate transcription in vitro. Different activators work with different combinations of TAFIIs to enhance transcription and all of them have TAFII 250 in common. This suggests that TAFII250 serve as an assembly factor around which other TAFs aggregate.

13. Exceptions to the Universality of TAFs and TBP
TAFs and TBP are not universally required for transcription of class II genes
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

14. 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

15. Structure of 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

16. Function of TFIIH 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
TFIIH is the last general transcription factor to join the preinitiation complex

17. 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

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

19. Figure 11.25

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. 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 - 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

24. TFIIS Stimulates Proofreading of Transcripts
TFIIS stimulates proofreading - 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

25. Class I Factors
RNA polymerase I and 2 transcription factors make up the preinitiation complex
Transcription factors:
A core-binding factor - SL1 or TIF-IB
A UPE-binding factor - upstream-binding factor (UBF) or upstream activating factor (UAF)

26. Class I Factors
SL1 is the fundamental transcription factor required to recruit RNA polymerase I
UPE-binding factor - is an assembly factor that helps SL1 to bind to the core promoter element

27. 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
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

28. This project is funded by a grant awarded under the President’s Community Based Job Training Grant as implemented by the U.S. Department of Labor’s Employment and Training Administration (CB ). NCC is an equal opportunity employer and does not discriminate on the following basis:
against any individual in the United States, on the basis of race, color, religion, sex, national origin, age disability, political affiliation or belief; and
against any beneficiary of programs financially assisted under Title I of the Workforce Investment Act of 1998 (WIA), on the basis of the beneficiary’s citizenship/status as a lawfully admitted immigrant authorized to work in the United States, or his or her participation in any WIA Title I-financially assisted program or activity.

29. Disclaimer
This workforce solution was funded by a grant awarded under the President’s Community-Based Job Training Grants as implemented by the U.S. Department of Labor’s Employment and Training Administration.  The solution was created by the grantee and does not necessarily reflect the official position of the U.S. Department of Labor.  The Department of Labor makes no guarantees, warranties, or assurances of any kind, express or implied, with respect to such information, including any information on linked sites and including, but not limited to, accuracy of the information or its completeness, timeliness, usefulness, adequacy, continued availability, or ownership.  This solution is copyrighted by the institution that created it.  Internal use by an organization and/or personal use by an individual for non-commercial purposes is permissible.  All other uses require the prior authorization of the copyright owner.