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

The Class II Preinitiation Complex

• Class II preinitiation complex contains: specific order– Polymerase II– 6 general transcription factors:

• TFIIA• TFIIB• TFIID• TFIIE• TFIIF• TFIIH

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

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

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

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

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

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

Transcription Enhancement by Activators

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)

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

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

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