Molecular BiologyFourth Edition

Chapter 11

General Transcription Factors in Eukaryotes

Lecture PowerPoint to accompany

Robert F. Weaver

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

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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

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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

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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

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Model of Formation of the DABPolF Complex

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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

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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

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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

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Model for the Interaction Between TBP and Promoters

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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

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Transcription Enhancement by Activators

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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

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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

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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

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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

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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

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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

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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

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Role of TFIIE and TFIIH

TFIIE and TFIIH

• Not essential for – Formation of an open promoter complex – Elongation

• Required for promoter clearance

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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

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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

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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

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Schematic Model

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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

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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

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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

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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

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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)

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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

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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

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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

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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

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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

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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

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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

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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

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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