II Transcription

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WS 13/14 Strukturbiologie, Transcription 1

TRANSCRIPTION

a) Transcription in prokaryotesb) Transcription in eukaryotes


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Fig.

Stryer

Transcription in prokaryotes and eukaryotes

The fundamental mechanism of transcription is conserved among cellular RNApolymerases, yet there are also marked differences between prokaryotes and eukaryotes:

Transcription and translation are coupled in bacteria; wherease transcription andtranslation are uncoupled in eukarya.

-Stages of Transcription: Initiation, Elongation, Termination-transcription bubble, unwound region of about 15 base pairs of the DNA template andsome eight residues of the RNA transcript hybridized with the DNA in the center of thebubble.


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Transcription cycle in prokaryotes

INITIATION of transcription1. Binding of polymeraseas aholoenzyme (sfactor plus corepolymerase)2. Open complex formation(transcription bubble).Unwinding of DNA, forming singlestrandedness within the active site.3. Initial RNA synthesis. Up to 10bp of RNA is synthesized. Duringthis initial step the polymerase isnot very efficient and can easily falloff.

www.bmb.psu.edu/courses/bmmb501/bmmb597a_fa

03/reese/16_lect_gene_reg_1_.pdf -

3 distinct phases:

INITIATION,ELONGATION,TERMINATION.


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Strukturbiologie, Transcription 5

a) Transcription in prokaryotes

In prokaryotes, transcription and translation are closely coupled.

Gene transcriptionis regulated by protein transcription factorsthat bind tooperator DNAand thus influence the ability of RNA polymerase to bind toa promoter region and initiate transcription.

Protein transcription factors are regulated by cellular environmental factors(e.g. transcription factors,allosteric effectors), which can include smallmolecules, another protein or metal ions. Transcription can be blocked bybinding of a specific repressor (e.g. lac) protein at a DNA site called anoperator. These DNA binding proteins recognize specific DNA sequences viadistinct DNA-binding domains.

Gene transcription in bacteria, Schreiter, 2007


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Transcription in prokaryotes - RNAP

Y.W. Yin and T.A. Steitz, Structural basis for the transition from initiation toelongation transcription in T7 RNA polymerase. Science298(2002).


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Transcription in prokaryotes

Crystal structures of the T.thermophi luselongationcomplex (ttEC) with thenon-hydrolysablesubstrate analogueAMPcPP (Vassylyev et al,nature 2007, 3)

Overall view of the ttEC/AMPcPPcomplex.

The DNA template, non-template andRNA strands are in red, blue and yellow,respectively.

The BH, the TH and the rest of the RNAPmolecule are in magenta, cyan andgrey, respectively.

The insertion and preinsertion NTPanalogues and Stl are designated bygreen, orange and black, respectively.

The catalytic Mg2+ions (MgI and MgII)are shown as magenta spheres. a, b,


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-Eukaryotes: Transcription and Translation are uncoupled

b) Transcription in eukaryotes

-Eukaryotes: 3 differentRNA polymerases (Pol I,Pol II, Pol III):

Regulatory elements ofeukaryotic transcription(TATA-box, -25)


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3D structure of the nucleosome

Surface representation of the histone

octamer

Structure of the nucleosome coreparticle; (14 independent DNA-bindinglocations)

Review, Karolin Luger

-In eukaryotes: Chromatin is composed of nucleosomes, which consist of an

octamer of histones around which 147 base pairs of DNA are wrapped.

Structure of the nucleosome, T. Richmond Lab, 1997


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Transcription in eukaryotes4 October 2006

The Royal Swedish Academy of Sciences has decided toaward the Nobel Prize in Chemistry for 2006 to

Roger D. Kornberg

Stanford University, CA, USA

"for his studies of the molecular basis of eukaryotictranscription".

Kornberg's contribution has culminated in his creation of detailedcrystallographic pictures describing the transcription apparatus in full

action in a eukaryotic cell. In his pictures (all of them created since 2000)we can see the new RNA-strand gradually developing, as well as the role ofseveral other molecules necessary for the transcription process. The picturesare so detailed that separate atoms can be distinguished and this makes itpossible to understand the mechanisms of transcription and how it isregulated.


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Structure determination of RNApolymerase II and complexes

1983 2-D protein crystals on lipid layers

1991 2-D crystals seed 3-D crystals (poor diffraction-work under

Argon)1998 Diffraction phased with heavy atom clusters

2000 Structure of RNA polII at 2.8 resolution

2002 Structure of transcribing complex 3.3

2002-ongoing Series of structures of transcribing complexs (2.9-4.4 ), complexes with bound inhibitors .....


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The Pol II transcription machinery (>3 MioDa)

Pol IIis capable of unwinding DNA, synthesizing RNA, and rewindingDNA. But Pol II alone is incapable of recognizing a promoter andinitiating transcription. For these essential functions, the participation

of the General Transc r ipt ion Factors is required. Mediator is co-activator, a co-repressor, and a general transcription factor all in one.Mediator, a megaDalton multiprotein complex, enables the regulationof transcription; it bridges between gene activator proteins atenhancers and RNA polymerase II (pol II) at promoters.

Pol II: DNA unwinding

RNA polymerization

proofreading

GTFs (TFIIB,-D,E,F,-H): promoter recognition

Mediator: interaction with activator proteinsand polII; essential for transcription


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Structure determination of the polymerase inthe form of a transcribing complex (3.3)

(B) Comparison of structures offree Pol II (top) and the Pol IItranscribing complex (bottom). Theclamp (yellow)closes on DNAand RNA, which are bound in thecleft above the active center. Theremainder of the protein is in gray.


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DNA can be seen entering thetranscribing complex in duplexform and unwinding three basesbefore the active site. Then thetemplate strand makes a sharpbend, and as a result, the nextbase is flipped, pointing downtowards the active site. This baseis paired with that of theribonucleotide just added to theRNA strand. The structure revealseight more DNA-RNA hybridbasepairs and one additional base onthe template DNA strand. Theremainder of the template strand,the RNA, and the nontemplateDNA strand are not seen, due to

motion or disorder.

Crystal structure of the Pol II transcribing complex

Gnatt et al, Science 2001


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Transcription Initiation mechanisms

How is straight duplex promoter DNA melted, bent, and inserted in thePol II active center, enabling the initiation of transcription?

Bushnell, D.A., et al. (2004) Structural basis of transcription: an RNA polymerase II-TFIIBcocrystal at 4.5 ngstrms. Science

These DNAtransactions aremade by the GTFsTFIIB , -D, -E, -F, and-H.

TFIIB stabilizes aninitial transcribingcomplex and the N-terminal regionforms Zn ribbon and

B finger.


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Transcription Initiation mechanisms

The structure of the Pol II-TFIIBcomplex revealed distinct functions ofthe N- and C-terminal domains of TFIIB. The N-terminal domain(yellow) begins with a Zn ribbonthat binds the Pol II surface adjacentto the clamp and wall.Then the polypeptidecontinues across thesaddle between the clamp and wall and plunges towards the active

center, from which it loops back and remerges across the saddle.


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The loop, termed the B finger, occupies almost the same locationas the DNA-RNA hybrid in a transcribing complex.Superimposing the B finger and the DNA-RNA hybrid from thetranscribing complex structure reveals no interference with thetemplate DNA strand or with the RNA up to position 5, but a stericclash with the RNA at positions 6 and beyond.


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B finger is not only compatiblewith a hybrid containing five

residues of RNA, but isrequired for stability of shortDNA-RNA complex (BiaCoreexperiments).

When the RNA grows beyond five or six residues, however, it mustcompete with TFIIB for space on the Pol IIsaddle. If TFIIB wins thecompetition, initiation is aborted and must be tried again. If the RNAwins, TFIIB is ejected and Pol II is released from the promoter tocontinue and complete transcription.

The B finger thus explains two crucial but for a long time mysteriousaspects of Pol II transcription, abortive initiation and promoter escape. Inthese respects, it resembles the sigma factor in bacterial transcription.


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Model of open promoter complex

Structure of an RNA polymerase II-TFIIB complex and the transcriptioninitiation mechanismScience, 2010, Kornberg Lab


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Initiation: Model of an RNA polymerase II-TBP-TFIIB-DNA complex

Structure of theC-terminal regionof TFIIB (pink)complexed withTBP (green) and

TATA-boxcontaining DNAwas docked tothe structure ofthe Pol II-TFIIBcomplex (clamp,

yellow), TFIIB-NT-region), wall(blue).


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Model of an RNA polymerase II-TBP-TFIIB-DNA complex

after addingstraight B-formDNA:

TATA-box-

saddle: 15bp;saddle-activesite: 12 bp

= ca 27 bp!!

distance TATA-box totranscription startsite in promoters25-30 bp


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Strukturbiologie, Transcription26

Docking a complex of a C-terminal TFIIB fragment, the TATA-bindingprotein (TBP) subunit of TFID, and a TATA box DNA fragment:

First, the DNA fit snugly against the protein:TBP evidentlyconfigures promoter DNA to the contours of the Pol II surface.

Second, the DNA downstream of the TATA box ran past the saddle.The distance from the TATA box to the saddle is about 1.5 turns of the

double helix, or 15 base pairs (bp).

We know from the transcribing complex structure that about 12residues are required to cross the saddle to the active site. Thesum of 15 bp from the TATA box and 12 residues to the active siteis 27

bp, closely coincident with the spacing of 2530 bp from the TATAbox to the transcription start site of almost all Pol II promoters. Inthis way, Pol II-TFIIB interaction may determine the location of thetranscription start site.


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Initiation: Transcription bubble (complex with TFIIF)

The structure includes a completetranscription bubblenot only thetemplate DNA strand withassociated RNA, but also thenontemplate DNA strand, and theregion upstream of the bubble

where duplex DNA is reformedfollowing transcription.yellow: TFIIF; green: coding DNA;red: RNA; cyan: template DNA

The interaction of thenontemplate strand with TFIIFmay trap a transient bubble inpromoter DNA, leading to theinitiation of transcription.


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Transcription initation

The structuresof Pol II, TBP, andTFIIB come fromX-raycrystallography.

The structures ofTFIIE, TFIIF, andTFIIH (helicase)are from electroncrystallographyand from cryo-

electronmicroscopy andsingle particleanalysis.


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Transcription initation - Complete minimal RNApolymerase II transcription initiation complex

TBPbends the promoter DNA aroundthe polymerase and the CTD of TFIIB.

The NTD of TFIIBbrings the DNA to apoint onthe polymerase surfacefromwhich it need only follow a straight path

and, by virtue of the conserved spacingfrom TATA box to transcription start sitein Pol II promoters, the start site isjuxtaposed with the active center.

TFIIE enters the complex and recruits

TFIIH, whose ATPase/helicasesubunitintroduces negativesuperhelical tension in the DNA.


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Transcription initation - Complete minimal RNApolymerase II transcription initiation complex

Thermal unwinding produces atransient bubble, which is capturedby TFIIFbinding to the nontemplatestrand. The DNAcan now bend in thesingle stranded region and descend intothe Pol II active center.Initiation and the synthesis of RNAensue, initially stabilized by the B finger.Synthesis of a transcript greater thanabout 6 residues in length leads to the

displacement of TFIIB, promoterescape, and the completion oftranscription.


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Other essential tasks of transcription:

Translocation

Nucleotide addition

Fidelity of Transcription

RNA escape

Regulationthe role of Mediator


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Translocation: Bridge helix might serve as molecular ratched

Straight and bent states of the bridge helix in RNA polymerase II (yeast) andbacterial RNA polymerase structures. The bend produces a movement of 3 inthe direction of the template strand, corresponding to one base pair step alongthe strand.


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A cycle of nucleotide addition by RNA polymerase II

At the upper left, thestructure of the

transcriping complex isshown, omitting all butthe DNA and RNA nearthe active center andthe bridge helix(green).The ribonucleotide inthe active center, justadded to the RNAchain, is yellow.At the lower leftis thestructure aftertranslocation of DNAand RNA across thePol II surface.


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At the lower rightisthe structure with anunmatched NTP inthe entry (E) site. Atthe upper rightis

the structure withNTP, matched forpairing to the codingbase in the templatestrand, in the

addition (A) site.


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All four NTPs were

seen to bind an entry orE site, whereas onlythe NTP correctlymatched for basepairing with the codingbase in the DNA was

seen to bind in theactive center, at thenucleotide addition orA site. The orientationof NTP in the E sitewas inverted with

respect to that in the Asite, leading to thesuggestion that NTPsin the E site rotate tosample base pairing inthe A site.


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Bridge helix update

Cheung et al, Structural basis of initial RNA polymerase II transcription, EMBOJ, 2011


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But 3D structure did not explain the fidelity of transcription: Theenergy of base pairing, through two or three hydrogen bonds to the

template DNA, is far less than required to account for the selectivity ofthe polymerase reaction.

2006:

New structures of RNA polymerase II (Pol II) transcribing complexesreveal a likely key to transcription. The trigger loopswings beneath acorrect nucleoside triphosphate (NTP) in the nucleotide addition site,closing off the active center, and forming an extensive network ofinteractions with the NTP base, sugar, phosphates, andadditional Pol II residues. A Hisside chain in the trigger loop,precisely positioned by these interactions, may literally trigger

phosphodiester bond formation. Recognition and catalysis arethus coupled, ensuring the fidelity of transcription.


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Fidelity of transcription: Trigger loop contacts NTP in the A site

Template DNARNA

Trigger Loop

NTP in A site(purine,pyrimidine NT)

The trigger loop

swings beneath acorrect nucleosidetriphosphate(NTP) in thenucleotide additionsite, closing off the

active center, andforming anextensivenetwork ofinteractions withthe NTP base,

sugar,phosphates, andadditional Pol IIresidues.


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Fidelity of transcription: Trigger loop contacts NTP in the A site

Template

DNARNA

TriggerLoop

NTP in A site(purine,pyrimidineNT)

A Hisside chain inthe trigger loop,precisely positionedby theseinteractions, may

literally triggerphosphodiesterbond formation.Recognition andcatalysis are thuscoupled, ensuring

the fidelity oftranscription.


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The trigger loop contacts allmoieties of the NTP - the base,the phosphates and through otherPol II residues, the sugar as well.The resulting network of

interactions even includes the 2-OH group of the nucleotide justadded to the end of the RNA.

The importance of these interactions is shown by mutations affectingtranscription.


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Trigger loop couples nucleotide selection to catalysis

Alignment of the trigger loopwith the NTP and theprecise positioning of ahistidine side chain, 3.5 from the -phosphate. Thehistidine promotes the flow

of electrons duringnucleophilic attack of the 3-OH at the chain terminusand phosphoanhydride bondbreakage. It serves as aproton donor for the

pyrophosphate leavinggroup. It literally triggersphosphodiester bondformation.

N l tid l ti b li t ith th t i


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Nucleotide selection by alignment with the triggerloop, coupling recognition to catalysis

The electronic transactions involved in trigger loop function require precise alignment of theinteracting moieties. This is achieved for a correct NTP by formation of the trigger loopnetwork. In the case of an incorrect NTP, for example a 2-deoxy NTP, misalignment isprofound. A double helix formed with a 2-deoxy nucleotide is 2 narrower than that formed

by a ribonucleotide.


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Separation of RNA transcript from the template- 3D structure in the posttranslocation state

Westover, K.D., et al. (2004) Structural basis of transcription:separation of RNA from DNA by RNA polymerase II. Science.

-7

-8

-9

-10

Forkloop

RudderLid

Release of RNA transcript

from DNA -RNA hybridrevealed in the structure ofan RNA polymerase IItranscribing complex. Theupstream end of the DNA -RNA hybrid helix, 7-10residues from the active

center, is shown on theleft, with distancesbetween the DNA and RNAbases indicated.The entireDNA -RNA hybrid helix isshown on the right, along

with protein loops involved inhelix melting (rudder and lid)and stabilization (fork loop).


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-7

-8

-9

-10

Forkloop

RudderLid

Base pair 7 of the DNA-RNA hybrid in thisstructure appears normalthe bases arecoplanar, with a distanceappropriate for hydrogen

bonding between them.Base pairs 8, 9, and 10,however, showincreasing deviations,and consequent splayingapart of the DNA and

RNA strands. The strandseparation is due to theintervention of threeprotein loops, termedfork loop 1, rudder, andlid.


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-7

-8

-9

-10

Forkloop

RudderLid

Rudder and lid liebetween DNA and RNA.Rudder contacts DNA,Lid RNA. A Phe sidechain of the lid serves aswedge to maintain

separation of the strands.Fork loop contacts thesugar-phosphatebackbone of the hybridhelix at base pairs 6 and7, stabilizing the helix,

preventing the DNA-RNAhybrid from unravelingfurther and inhibitingtranscription.


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Transcriptionregulation:the role ofMediator

Mediator is a keyregulator of eukaryotictranscription,connecting activatorsand repressors boundto regulatory DNAelements with Pol II.


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Transcription regulation:the role of Mediator

Cryo-EM structure, 35 resolution, Asturias Lab2002; Extension of the structure to atomicresolution will one day reveal the regulatorymechanism

In the yeast Saccharomycescerevisiae, Mediator comprises

25 subunits with a total mass ofmore than one megadalton and isorganized into three modules,called head, middle/arm and tail.

Architecture of the Mediator head module, nature2011; x-ray structure of mediator head; 4.3 A


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Transcription regulation:the role of Mediator

In the yeast Saccharomyces cerevisiae, Mediator comprises 25 subunits with atotal mass of more than one megadalton and is organized into three modules,

called head, middle/arm and tail.

Structure of the Mediator head module:

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II Transcription