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TF. TBP. TATA. Promoter. GTFs and PIC assembly. TBP. TFIIB. TFIIA. TFIIE. TFIIF. TFIIH. GTFs and PIC assembly. General transcription factors (GTFs) make RNAPII capable of selective initiation in vitro Highly conserved RNAPII+GTFs = ca. 30 polypeptides ≈ 2 MDa. +. =. PIC.

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Gtfs and pic assembly

TF

TBP

TATA

Promoter

GTFs and PIC assembly


Gtfs and pic assembly1

TBP

TFIIB

TFIIA

TFIIE

TFIIF

TFIIH

GTFs and PIC assembly

  • General transcription factors (GTFs)

    • make RNAPII capable of selective initiation in vitro

  • Highly conserved

  • RNAPII+GTFs = ca. 30 polypeptides

    • ≈ 2 MDa

+

=

PIC

Correct

initiation

of trx

in vitro


Linear assembly of pic the preinitiation complex
Linear assembly of PIC -the preinitiation complex

  • Nucleation

    • TFIID+TATA form an “initial committed complex”

    • TAFs + INR may also initiate PIC-assembly

    • Common: a core sequence is recognized by a seq.spes.GTF

  • Link

    • initial complex recognized by TFIIB

    • With TFIIB bound, the complex becomes accessible to RNAPII

  • RNAPII recruitment

    • Assembly of RNAPII assisted by TFIIF

    • Minimal initiation complex formed

  • Maturationto complete trx competent PIC

    • Minimal initiation complex (DABF-pol) NOT trx.competent

    • Recruitment of TFIIH and TFIIE necessary

    • This step is unique for RNAPII


  • Alternatives to linear pic assembly
    Alternatives to linear PIC-assembly

    Alternative

    Nucleation events

    Nucleation

    Link

    RNAPII recruitment

    Holoenzyme

    2-step

    alternative

    Maturation


    Tbp tfiid function
    TBP [TFIID] function

    • Binds TATA - main sequence recognition event during PIC assembly

      • Binds a variety of different TATA-like sequences

      • A slow binding reaction

      • minor groove contact

      • binds as monomer

    • Affinity of TBP for TATA contributes to promoter strength

    • Binds also several other polypeptides

      • activators (Sp1, Tax1, E1A)

      • TAFs (dTAF110, dTAF40)

      • GTFs (TFIIB, TFIIA)

      • inhibitors

    • TBP = universal TF involved in all three pol syst.

      • TBP i SL1, TFIID, TFIIIB

    Other factors

    N

    DNA


    Tbp versus tfiid

    TAFs

    TBP

    TBP versus TFIID

    • Subunit-structure

      • TFIID = TBP + multiple TAFs

      • mammalian TFIID: 750 kDa (II), 300 kDa (III) and 200 kDa (I)

      • TBP only a small core in the TFIID complex

        • human 38 kDa, yeast 27 kDa, Arabidopsis 22 kDa

      • TBP = N-term divergent domain + C-term. conserved domain

        • C-term domain 180aa symmetric

          • Carries all essential functions

        • N-term domain divergent

          • probably involved in regulating DNA binding

    TBP TFIID

    N


    Tbps saddle structure
    TBPs saddle-structure

    Convex

    surface

    protein

    Concave

    inside

    DNA

    Stigbøyler

    stirrups

    • 3D: saddle-structure

      • Twofold symmetry -form of a saddle.

      • Concave inside binds DNA in minor groove through a 10-stranded antiparallel -sheet

      • Convex surface binds other GTFs via 4 -helixes

      • loop (“stirrup”) on each side with Phe side-chains intercalating in DNA


    Tbps effect on dna

    .. but this way

    Not like this

    TBPs effect on DNA

    • DNA-structure is distorted upon TBP binding

      • DNA severely bended, unwinded and distorted

      • DNA shaped by TBP´s -sheet

      • The intercalating Phe-residues contributes to kink

    • Effect?

      • Upstream and downstream elements brought closer together

      • incompatible with nucleosome structure


    A two step mechanism of tbp binding to dna
    A Two-Step Mechanism of TBP Binding to DNA

    • First step

      • Full-length TBPWT first binds to TATA box to form an unbent TBP-TATA box complex.

    • Second step

      • Then, this unbent complex slowly forms the bent TBP-TATA box complex.

      • TFIIB can directly recognize the unbent and/or bent TBP-TATA-complexes to form the bent TBP-TATA box complex.

      • Does this two-step mechanism apply within the context of TFIID?


    Tfiib
    TFIIB

    • Functions in PIC-assembly as adaptor - a molecular bridge that couples TBP-TATA with RNAPII

      • TFIIB recognizes the distorted TBP-TATA complex

        • contacts DNA on both sides of TBP-TATA

        • upstream via major groove (BRE) and downstream via minor groove

        • Provides directionality to the complex through assymmetric binding

      • TFIIB mediates RNAPII binding

        • interaction also with TFIIF

    • Function in initiation: “Measures” distance TATA - TSS

    TATA

    BRE

    TFIIB

    +1

    TSS


    Tfiib1
    TFIIB

    • TFIIB also contact point for activators

      • VP16, Steroid hormone receptorer, fushi tarazu, TAF40

    • TFIIB-BRE: a repressive interaction?

      • The BRE was recently reported to repress basal transcription, with activator-mediated disruption of the BRE-TFIIB interaction as a proposed mechanism of gene activation.

    BRE

    TFIIB

    +1


    Tfiib structure
    TFIIB-structure

    • C-terminal core domain (cTFIIB)

      • C-term core with 2 imperfect direct repeats (2x 75aa) that binds TBP-TATA complex

      • each repeat = 5 -helices  compact globular domain (cyclin A-like)

      • HTH motiv that binds BRE (not conserved in yeast and plants)

      • DNA-contact before and after TBP

    • N-terminal (nTFIIB) essential for RNAPII contact

      • cysteine-rich region that forms a “zinc-ribbon” + B-finger

      • mediate contact wtih RNAPII-TFIIF complex through a penetration mechanism

    C-term core

    TFIIB

    +1

    N


    Tfiibc structure
    TFIIBc structure

    TBP

    Two globular repeats

    contact DNA

    before and after TBP

    TSS

    TFIIB


    Zn ribbon b finger bridge to rnapii
    Zn-ribbon + B-finger = bridge to RNAPII


    Tfiib links tata and rnapii and penetrates the active site
    TFIIB links TATA and RNAPIIand penetrates the active site

    TBP

    BC link

    TATA-pol

    TFIIB

    The N-terminal domain of TFIIB (Zn ribbon) binds the dock domain, where its B-finger plunges down into the RNAPII active center, loops back and remerges across the saddle.

    BN active

    site


    Tfiib b finger penetrates rnapii
    TFIIB-B-finger penetrates RNAPII

    Boeger, H., Bushnell, D.A., Davis, R., Griesenbeck, J., Lorch, Y., Strattan, J.S., Westover, K.D. and Kornberg, R.D. (2005)

    Structural basis of eukaryotic gene transcription. FEBS Lett, 579, 899-903.


    Tfiib b finger takes the place of rna expelled when trx starts
    TFIIB-B-finger takes the place of RNAExpelled when trx starts

    B finger occupies the same location as the DNA–RNA hybrid.

    TFIIB may enhance the formation of an early transcribing complex before a length of 9 bp, required for optimal stability, is attained.

    As RNA grows, RNA and TFIIB must compete for space. If RNA wins, TFIIB is ejected and the pol is released from the promoter to complete trx of the gene. If TFIIB wins, initiation aborts and must be tried again.

    The B finger thus explains abortive initiation and promoter escape.

    TBP

    BC link

    TATA-pol

    TFIIB

    BN active

    site



    Tfiia
    TFIIA Complex

    • Controversial

      • not essensial in vitro with TBP and purified components

      • required with TFIID and less purified system

    • Function

      • counteracts repressors associated with TFIID (Dr1, topoI, MOT1)

      • Stabilizes the TBP-TFIIB complex

        • TFIIA needed for binding of TBP to nucleosomal TATA

      • TFIIA is able to enter the PIC assembly on all steps after TFIID binding

      • Required for activator-response


    Structure of tfiia
    Structure of TFIIA Complex

    • human/drosophila heterotrimer: 37 + 19 + 13 kDa ()

      • Both  and  product of the same gene - the ab precursor is cleaved to a + b

    • yeast: heterodimer: 32 + 13 kDa

      • TOA1 32kDa (homologous to human  and ) essensial

      • TOA2 13 kDa essensial

    • Antirepression requires 

    • Activation requires 

    • 3D  two domains form an L-formed structure

      • TOA1 and TOA2 intertwined

      • Both C-terminals generate a compact -sheet ( -sandwich,  -barrel)

      • Both N-terminals generate a “four-helix bundle”

    Yeast TOA1

    Human a

    Human ß

    L

    C

    N


    Tfiia structure
    TFIIA structure Complex

    C-terminal ß-barrel

    contacts DNA and TBP

    TFIIA

    N-terminal

    4-helix bundle.

    Probably activator contact


    Tfiia structure1
    TFIIA structure Complex

    TBP

    C-terminal ß-barrel

    contacts DNA and TBP

    TFIIA

    N-terminal

    4-helix bundle.

    Probably Activator contact


    Yeast tfiia tbp dna
    Yeast TFIIA + TBP + DNA Complex

    TBP

    TFIIA


    Tfiia dna interaction
    TFIIA - DNA-interaction Complex

    • Interaction with DNA upstream TATA

    • C-terminal -barrel  both TBP- and DNA-interaction

      • TBP-TFIIA: the edges of the two -structures interact  extended -sheet

      • DNA-TFIIA: C-terminal -barrel contacts phosphates 3 bp upstream TATA

      • Explains why TFIIA stabilizes TBP-DNA complex

    • TFIIAs N-terminal -helix structure generates an interaction domain necessary for activator contact

      • Rational explanation of:

      • Antirepression requires  which generate -barrel with TBP+DNA contact

      • Activation requires  which also generate the N-terminal interaction domain

    • TFIIA and TFIIB bind on opposite sides of DNA without collision

    • TBPs convex surface still exposed for other interactions


    Tfiia tbp tfiib place for all
    TFIIA-TBP-TFIIB: place for all Complex

    TBP

    TFIIA

    TFIIB


    Tfiif also called rap rnapii ass faktor
    TFIIF Complex (also called RAP = RNAPII-ass. faktor)

    • Structure:

      • Heterodimer in higher eukaryotes: RAP30 + RAP74 (Mw: 26 + 58 kDa)

      • S.cer.TFIIF heterotrimer: 105, 54, 30 kDa

    • Distinct feature: function in initiation and elongation

    • Initiation - helps in the recruitment of RNAPII

      • Stable association of RNAPII requires TFIIF

      • TFIIF-TFIIB associate in solution

      • TFIIF-RNAPII associate in solution

    • Initiation: a role in recruitment of TFIIE+TFIIH

    • Elongation: enhances catalytic velocity of RNAPII

      • More later

    TFIIB

    TFIIF


    Tfiif heterotetramer rap30 2 rap74 2
    TFIIF Complex = heterotetramer (RAP302 RAP742)

    RNAPII

    • RAP30: Two -related domains

    • RAP74:

      • Required for stimulation of elongation

      • RAP74 is strongly phosphorylated in vivo

        • Kinase? Possibly TAFII250

        • TFIIF becomes more active when phosphorylated

    30

    30

    DNA

    TFIIB

    74

    74

    P

    P

    P

    P

    DNA

    RNAPII


    Tfiif dna contacts
    TFIIF DNA-contacts Complex

    • Complex pattern of protein-DNA contacts

    • Explained by wrapping of DNA around RNAPII-TFIIF?

    74

    74

    30

    30

    ?

    TATA

    INR


    3d of tfiif
    3D of TFIIF Complex

    • TFIIF (blue) is distributed across the surface of the polymerase.

    • The distribution of the second largest subunit of TFIIF was very similar to the sigmal subunit of bacterial RNA polymerase.



    Tfiie
    TFIIE Complex

    TFIIEb

    • Structure

      • heterotetramer 22: 56 + 34 kDa

      • Contacts DNA in and just downstream of trx bubble

    • Function in trx.initiation

      • Recruitment of TFIIH to PIC

      • Regulates the activity of TFIIH

    • Role in NER (nucleotide excision repair)

      • Damage recognized by XPA

      • XPA binds TFIIE

      • TFIIE recruits TFIIH

      • Repairosome is formed

    34

    34

    56

    56

    TFIIEa


    Tfiih
    TFIIH Complex

    • The most complex of the GTFs - 9 subunits

      • Alle tilhørende gener essensielle

    • The only GTF with enzymatic activity:

      • Two Helicases (ATP-dep.)

      • [ATPase (DNA-dep.)]

      • CTD-kinase

    • Kinase substrat:

      • CTD - preferred substrate of Holo TFIIH

      • GTFs

        • TBP

        • TFIIEa

        • TFIIFa (RAP74)

      • Andre TFs

        • Oct, p53, RARa, ERa, pRb



    Tfiih structure1

    core Complex

    kinase

    TFIIH-structure

    Helicases utilise the energy of nucleotide hydrolysis

    to unwind nucleic acid duplexes.

    NER - nucleotide excision repair

    Surprising

    Link to

    DNA-repair

    • Multisubunit factor( human / yeast )

      • 89 kDa XPB / SSL2 (p105) NER-function ATPase/3´-5´-helicase

        • NTP-site mutated  lethal + trx.dead

        • XPB-helicase is necessary for trx.activity

        • Explains ATP requirement in initiation of trx

      • 80 kDa XPD/ RAD3 (p85) NER-function ATPase/5´-3´-helicase

        • NTP-site mutated  not lethal + trx.OK + NER-defect

        • XPD-helicase not required for trx. activity

      • 62 kDa P62 / TFB1 (p75) UV-hypersens.

      • 50 kDa P52/ TFB2 (p55)

      • 44 kDa P44 / SSL1 (p50) (supr. of stem-loop) zinc finger motif

      • 34 kDa P34 / TFB4 (p37) zinc finger motif

      • 32 kDa MAT1 / TFB3 (p38) ring finger motif, cdk-assembly factor

      • 38 kDa cyclin H / CCL1 (p45+p47) cyclin-partner for CDK7/MO15 and Kin28

      • 40 kDa CDK7, MO15 / KIN28 (p32) cyclin-dependent kinase

    • TFIIH dual function: in transcription initiat. and in NER


    Holo tfiih core tfiih cak linked by xpd

    Core TFIIH Complex

    Kinase

    (CAK)

    CAK

    Holo TFIIH = core TFIIH + CAK linked by XPD

    Bridge


    Tfiih multiple functions
    TFIIH multiple functions Complex

    • Function 1: promoter-melting assisted by helicases (2 steps, see below)

      • Model: 3´-5´-helicase + 5´-3´-helicase + ATP  chain separation around TSS

      • ATP-depent step in initation (in addition to CTD phosphorylation)

    • Function 2: CTD-kinase, role in promoter clearance

      • Modell: CTD-phosphorylation after chain separation and initiation  PIC disrupted  elongation complex leaves the promoter

    • Function 3: role in elongation

      • Model: TFIIH-kinase+ ATP  maintains hyperphosphorylated pol.II (counteracting the CTD phosphatase)

    • Function 4: role in DNA-repair (NER)

      • 5 of 9 subunits of TFIIH with a double function in trx.+repair

      • actively trx.genes are preferentially repaired

      • TFIIH can complement NER-deficient extract


    Assists in formation of open complex and promoter escape
    Assists in formation of open complex and promoter escape Complex

    1. ATP-dependent promoter melting - chain separation - open trx. complex

    2. ATP-dependent structural transition into an escape- competent conformation

    TFIIH helicase

    TFIIH helicase


    Tfiih also linked to the cell cycle
    TFIIH: also linked to the cell cycle? Complex

    • The TFIIH kinase = CAK = cdk7 + cyclin H + MAT-1

      • CAK = CDK activating kinase

      • CAK activates other cdk´s through Thr-phosphorylation

      • MAT-1 (a ring-finger protein) makes CAK constitutively active (Thr-indep.)

    • An open question :

      • Is CTD-phosphorylation regulated by the cell cycle?

    • Different answers :

      • No - probably not!

        • Argument : only 20% of all CAK in the cell is TFIIH-associated

        • S.cer. Has separate CAKs for TFIIH and cell cycle

        • Activity and level of CDK7, cyclin H and Mat1 do not change during cell cycle

      • Yes - May wll be!

        • TFIIH inhibited during mitosis concomitant with inhibition of CDK7 (CDC2-induced)

        • Cell cycle inhibitor INK4 inhibits CTD phosphorylation by CDK7

        • CDK8 can negatively regulate CDK7


    Model
    Model Complex

    Repair-

    proteins

    DNA repair

    Transcription

    CAK

    CAK

    Core TFIIH

    Core TFIIH

    CAK

    Cell cycle



    Pic assembly a gradual wrapping process

    TBP Complex

    TBP

    TFIIB

    RNAPII

    TBP

    TFIIB

    RNAPII

    TFIIF

    TFIIE

    PIC assembly - a gradual wrapping process?



    The trx cycle

    The trx cycle Complex



    Multiplicity of gtfs

    Multiplicity of GTFs? Complex

    Are a single set of GTFs universally used?

    …equally at all promoters?


    Several gtf complexes possible
    Several GTF complexes possible Complex

    • Several GTFs encoded by single copy genes

      • TFIIB, E, F, and H

      • Also true for RNAPII

    • However, multiple genes exist for specific GTFs

      • Multiple TFIIA related

      • Multiple TFIID related

      • Gene-selective developmental roles?

    • Consequence: several possible complexes possible

      • By replacing ”normal” versions with specific ones

      • By generating variant combinations of GTF-containing complexes


    Variant tbps trfs tbp related factors
    Variant TBPs: ComplexTRFs = TBP related factors

    ≥2 TBP like proteins

    in multicellular organisms

    TBP top view

    Drosophila

    TRF1

    TBP

    TRF2

    TLP

    TLF

    TRF

    TRP

    TBP bottom view

    • TRF1 - major part of TFIIIB, a RNAPIII factor

    • TRF1 binds pref TC-box (TTTTCT) in the core promoter of the Drosophila tudor gene, a direct target

    TBP specific

    TRF2 specific


    A diversity of complexes
    A diversity of complexes Complex

    • Many TBP complexes

    • Alternative TAF-containing complexes

    • Variant TFIIAs


    A diversity of core promoters may assemble gene specific complexes
    A diversity of core promoters may assemble gene-specific complexes

    • TATA core promoters require TBP, but not necessarily TAFs

    • Inr ± DPE core promoters require TAFs and hence indirectly TBP associated

    • TLF-dependent core promoters do not require TBP


    Diversity of core promoters

    Enormous complexes

    diversity

    Diversity of core promoters

    • GTF machinery shows some diversity

    • Activators and repressors (Tfs) show enormous diversity

    • Not thousands to one, but thousands to several

    Some

    diversity