General Transcription Factors in Eukaryotes Chapter 11

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

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

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 = 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

Model of Formation of the DABPolF Complex

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.

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

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.

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 11.25 – page 301

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

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.

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.

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

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

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

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

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

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

Figure 11.25

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

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

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

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

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

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)

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

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

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-15-162-06-60). 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.

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.