TRANSCRIPTION a) Transcription in prokaryotes b) Transcription in eukaryotes

TRANSCRIPTION a) Transcription in prokaryotes b) Transcription in eukaryotes WS 13/14 Strukturbiologie, Transcription 1

Transcription in prokaryotes and eukaryotes Fig. Stryer The fundamental mechanism of transcription is conserved among cellular RNA polymerases, yet there are also marked differences between prokaryotes and eukaryotes: Transcription and translation are coupled in bacteria; wherease transcription and translation are uncoupled in eukarya. -Stages of Transcription: Initiation, Elongation, Termination - transcription bubble, unwound region of about 15 base pairs of the DNA template and some eight residues of the RNA transcript hybridized with the DNA in the center of the bubble. 2

Transcription cycle in prokaryotes 3 distinct phases: INITIATION, ELONGATION, TERMINATION. INITIATION of transcription 1. Binding of polymerase as a holoenzyme (s factor plus core polymerase) 2. Open complex formation (transcription bubble ). Unwinding of DNA, forming single strandedness within the active site. 3. Initial RNA synthesis. Up to 10 bp of RNA is synthesized. During this initial step the polymerase is not very efficient and can easily fall off. www.bmb.psu.edu/courses/bmmb501/bmmb597a_fa 03/reese/16_lect_gene_reg_1_.pdf - 3

Transcription cycle in prokaryotes 4. Release of the s factor and synthesis beyond 10 bp of RNA (transition from initiation to elongation Durniak, K.J., Bailey, S., Steitz, T.A. (2008) The structure of a transcribing T7 RNA polymerase in transition from initiation to elongation Science). A structural change in the polymerase occurs. The jaws tighten down on the DNA and the elongation complex is MUCH more stable. 5. Highly processive ELONGATION phase 6. TERMINATION. A termination signal in the DNA forms a RNA hairpin in the emerging transcript. Its binding to polymerase stimulates a change in conformation (opening of the jaws ) and release. www.bmb.psu.edu/courses/bmmb501/bmmb597a_fa 03/reese/16_lect_gene_reg_1_.pdf - 4

a) Transcription in prokaryotes In prokaryotes, transcription and translation are closely coupled. Gene transcription is regulated by protein transcription factors that bind to operator DNA and thus influence the ability of RNA polymerase to bind to a promoter region and initiate transcription. Protein transcription factors are regulated by cellular environmental factors (e.g. transcription factors, allosteric effectors), which can include small molecules, another protein or metal ions. Transcription can be blocked by binding of a specific repressor (e.g. lac) protein at a DNA site called an operator. These DNA binding proteins recognize specific DNA sequences via distinct DNA-binding domains. Gene transcription in bacteria, Schreiter, 2007 Strukturbiologie, Transcription 5

Transcription in prokaryotes - RNAP Y.W. Yin and T.A. Steitz, Structural basis for the transition from initiation to elongation transcription in T7 RNA polymerase. Science 298 (2002). Strukturbiologie, Transcription 7

Transcription in prokaryotes Crystal structures of the T. thermophilus elongation complex (ttec) with the non-hydrolysable substrate analogue AMPcPP (Vassylyev et al, nature 2007, 3Å) Overall view of the ttec/ampcpp complex. The DNA template, non-template and RNA strands are in red, blue and yellow, respectively. The BH, the TH and the rest of the RNAP molecule are in magenta, cyan and grey, respectively. The insertion and preinsertion NTP analogues and Stl are designated by green, orange and black, respectively. The catalytic Mg 2+ ions (MgI and MgII) are shown as magenta spheres. a, b, Strukturbiologie, Transcription 8

b) Transcription in eukaryotes -Eukaryotes: Transcription and Translation are uncoupled -Eukaryotes: 3 different RNA polymerases (Pol I, Pol II, Pol III): Regulatory elements of eukaryotic transcription (TATA-box, -25) 9

3D structure of the nucleosome -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 Surface representation of the histone octamer Review, Karolin Luger Structure of the nucleosome core particle; (14 independent DNA-binding locations) Strukturbiologie, Transcription 10

Transcription in eukaryotes 4 October 2006 The Royal Swedish Academy of Sciences has decided to award the Nobel Prize in Chemistry for 2006 to Roger D. Kornberg Stanford University, CA, USA "for his studies of the molecular basis of eukaryotic transcription". Kornberg's contribution has culminated in his creation of detailed crystallographic 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 of several other molecules necessary for the transcription process. The pictures are so detailed that separate atoms can be distinguished and this makes it possible to understand the mechanisms of transcription and how it is regulated. Strukturbiologie, Transcription 12

Structure determination of RNA polymerase 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 Å resolution 2002 Structure of transcribing complex 3.3 Å 2002-ongoing Series of structures of transcribing complexs (2.9-4.4 Å), complexes with bound inhibitors... Strukturbiologie, Transcription 13

The Pol II transcription machinery (>3 MioDa) Pol II: GTFs (TFIIB,-D,E,F,-H): Mediator: DNA unwinding RNA polymerization proofreading promoter recognition interaction with activator proteins and polii; essential for transcription Pol II is capable of unwinding DNA, synthesizing RNA, and rewinding DNA. But Pol II alone is incapable of recognizing a promoter and initiating transcription. For these essential functions, the participation of the General Transcription Factors is required. Mediator is coactivator, a co-repressor, and a general transcription factor all in one. Mediator, a megadalton multiprotein complex, enables the regulation of transcription; it bridges between gene activator proteins at enhancers and RNA polymerase II (pol II) at promoters. 14

Structure determination of the polymerase in the form of a transcribing complex (3.3Å) (B) Comparison of structures of free Pol II (top) and the Pol II transcribing complex (bottom). The clamp (yellow) closes on DNA and RNA, which are bound in the cleft above the active center. The remainder of the protein is in gray. Strukturbiologie, Transcription 15

Crystal structure of the Pol II transcribing complex Gnatt et al, Science 2001 DNA can be seen entering the transcribing complex in duplex form and unwinding three bases before the active site. Then the template strand makes a sharp bend, and as a result, the next base is flipped, pointing down towards the active site. This base is paired with that of the ribonucleotide just added to the RNA strand. The structure reveals eight more DNA-RNA hybrid base pairs and one additional base on the template DNA strand. The remainder of the template strand, the RNA, and the nontemplate DNA strand are not seen, due to motion or disorder. 16

Transcription Initiation mechanisms How is straight duplex promoter DNA melted, bent, and inserted in the Pol II active center, enabling the initiation of transcription? These DNA transactions are made by the GTF s TFIIB, -D, -E, -F, and -H. TFIIB stabilizes an initial transcribing complex and the N- terminal region forms Zn ribbon and B finger. Bushnell, D.A., et al. (2004) Structural basis of transcription: an RNA polymerase II-TFIIB cocrystal at 4.5 Ångströms. Science Strukturbiologie, Transcription 17

Transcription Initiation mechanisms The structure of the Pol II-TFIIB complex revealed distinct functions of the N- and C-terminal domains of TFIIB. The N-terminal domain (yellow) begins with a Zn ribbon that binds the Pol II surface adjacent to the clamp and wall.then the polypeptide continues across the saddle between the clamp and wall and plunges towards the active center, from which it loops back and remerges across the saddle. 18

The loop, termed the B finger, occupies almost the same location as the DNA-RNA hybrid in a transcribing complex. Superimposing the B finger and the DNA-RNA hybrid from the transcribing complex structure reveals no interference with the template DNA strand or with the RNA up to position 5, but a steric clash with the RNA at positions 6 and beyond. 19

B finger is not only compatible with a hybrid containing five residues of RNA, but is required for stability of short DNA-RNA complex (BiaCore experiments). When the RNA grows beyond five or six residues, however, it must compete with TFIIB for space on the Pol II saddle. If TFIIB wins the competition, initiation is aborted and must be tried again. If the RNA wins, TFIIB is ejected and Pol II is released from the promoter to continue and complete transcription. The B finger thus explains two crucial but for a long time mysterious aspects of Pol II transcription, abortive initiation and promoter escape. In these respects, it resembles the sigma factor in bacterial transcription. Strukturbiologie, Transcription 20

Model of open promoter complex Structure of an RNA polymerase II-TFIIB complex and the transcription initiation mechanism Science, 2010, Kornberg Lab 21

Transcription Initiation TATA box binding protein (TBP) TBP: highly conserved CTD (180 aa); non-conserved NTD Sigler, Burley, 1993 TBP forms a saddle shaped molecule with stirrups. A b-sheet in TBP forms the DNA-binding site. TBP binds in the minor groove (strong hydrophobic interaction, H-bonds) and induces large structural changes in DNA (DNA helices on both sides form an angle of ca. 110º. Strukturbiologie, Transcription 23

Initiation: Model of an RNA polymerase II-TBP-TFIIB-DNA complex Structure of the C-terminal region of TFIIB (pink) complexed with TBP (green) and TATA-box containing DNA was docked to the structure of the Pol II-TFIIB complex (clamp, yellow), TFIIB- NT-region), wall (blue). 24

Model of an RNA polymerase II-TBP-TFIIB-DNA complex after adding straight B-form DNA: TATA-boxsaddle: 15bp; saddle-active site: 12 bp = ca 27 bp!! distance TATAbox to transcription start site in promoters 25-30 bp 25

Docking a complex of a C-terminal TFIIB fragment, the TATA-binding protein (TBP) subunit of TFID, and a TATA box DNA fragment: First, the DNA fit snugly against the protein: TBP evidently configures 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 12 residues are required to cross the saddle to the active site. The sum of 15 bp from the TATA box and 12 residues to the active site is 27 bp, closely coincident with the spacing of 25 30 bp from the TATA box to the transcription start site of almost all Pol II promoters. In this way, Pol II-TFIIB interaction may determine the location of the transcription start site. Strukturbiologie, Transcription 26

Initiation: Transcription bubble (complex with TFIIF) The interaction of the nontemplate strand with TFIIF may trap a transient bubble in promoter DNA, leading to the initiation of transcription. The structure includes a complete transcription bubble not only the template DNA strand with associated RNA, but also the nontemplate DNA strand, and the region upstream of the bubble where duplex DNA is reformed following transcription. yellow: TFIIF; green: coding DNA; red: RNA; cyan: template DNA Strukturbiologie, Transcription 27

Transcription initation The structures of Pol II, TBP, and TFIIB come from X-ray crystallography. The structures of TFIIE, TFIIF, and TFIIH (helicase) are from electron crystallography and from cryoelectron microscopy and single particle analysis. Strukturbiologie, Transcription 28

Transcription initation - Complete minimal RNA polymerase II transcription initiation complex TBP bends the promoter DNA around the polymerase and the CTD of TFIIB. The NTD of TFIIB brings the DNA to a point on the polymerase surface from which it need only follow a straight path and, by virtue of the conserved spacing from TATA box to transcription start site in Pol II promoters, the start site is juxtaposed with the active center. TFIIE enters the complex and recruits TFIIH, whose ATPase/helicase subunit introduces negative superhelical tension in the DNA. Strukturbiologie, Transcription 29

Transcription initation - Complete minimal RNA polymerase II transcription initiation complex Thermal unwinding produces a transient bubble, which is captured by TFIIF binding to the nontemplate strand. The DNA can now bend in the single stranded region and descend into the Pol II active center. Initiation and the synthesis of RNA ensue, initially stabilized by the B finger. Synthesis of a transcript greater than about 6 residues in length leads to the displacement of TFIIB, promoter escape, and the completion of transcription. Strukturbiologie, Transcription 30

Strukturbiologie, Transcription 32

Other essential tasks of transcription: Translocation Nucleotide addition Fidelity of Transcription RNA escape Regulation the role of Mediator Strukturbiologie, Transcription 33

Translocation: Bridge helix might serve as molecular ratched Straight and bent states of the bridge helix in RNA polymerase II (yeast) and bacterial RNA polymerase structures. The bend produces a movement of 3 Å in the direction of the template strand, corresponding to one base pair step along the strand. Strukturbiologie, Transcription 34

A cycle of nucleotide addition by RNA polymerase II At the upper left, the structure of the transcriping complex is shown, omitting all but the DNA and RNA near the active center and the bridge helix (green). The ribonucleotide in the active center, just added to the RNA chain, is yellow. At the lower left is the structure after translocation of DNA and RNA across the Pol II surface. Strukturbiologie, Transcription 35

A cycle of nucleotide addition by RNA polymerase II At the lower right is the structure with an unmatched NTP in the entry (E) site. At the upper right is the structure with NTP, matched for pairing to the coding base in the template strand, in the addition (A) site. Strukturbiologie, Transcription 36

A cycle of nucleotide addition by RNA polymerase II All four NTPs were seen to bind an entry or E site, whereas only the NTP correctly matched for base pairing with the coding base in the DNA was seen to bind in the active center, at the nucleotide addition or A site. The orientation of NTP in the E site was inverted with respect to that in the A site, leading to the suggestion that NTPs in the E site rotate to sample base pairing in the A site. Strukturbiologie, Transcription 37

Bridge helix update Cheung et al, Structural basis of initial RNA polymerase II transcription, EMBO J, 2011 Strukturbiologie, Transcription 38

But 3D structure did not explain the fidelity of transcription: The energy of base pairing, through two or three hydrogen bonds to the template DNA, is far less than required to account for the selectivity of the polymerase reaction. 2006: New structures of RNA polymerase II (Pol II) transcribing complexes reveal a likely key to transcription. The trigger loop swings beneath a correct nucleoside triphosphate (NTP) in the nucleotide addition site, closing off the active center, and forming an extensive network of interactions with the NTP base, sugar, phosphates, and additional Pol II residues. A His side chain in the trigger loop, precisely positioned by these interactions, may literally trigger phosphodiester bond formation. Recognition and catalysis are thus coupled, ensuring the fidelity of transcription. Strukturbiologie, Transcription 40

Fidelity of transcription: Trigger loop contacts NTP in the A site RNA NTP in A site (purine, pyrimidine NT) Trigger Loop Template DNA The trigger loop swings beneath a correct nucleoside triphosphate (NTP) in the nucleotide addition site, closing off the active center, and forming an extensive network of interactions with the NTP base, sugar, phosphates, and additional Pol II residues. Strukturbiologie, Transcription 41

Fidelity of transcription: Trigger loop contacts NTP in the A site RNA NTP in A site (purine, pyrimidine NT) Trigger Loop Template DNA A His side chain in the trigger loop, precisely positioned by these interactions, may literally trigger phosphodiester bond formation. Recognition and catalysis are thus coupled, ensuring the fidelity of transcription. Strukturbiologie, Transcription 42

The trigger loop contacts all moieties of the NTP - the base, the phosphates and through other Pol II residues, the sugar as well. The resulting network of interactions even includes the 2 - OH group of the nucleotide just added to the end of the RNA. The importance of these interactions is shown by mutations affecting transcription. Strukturbiologie, Transcription 43

Trigger loop couples nucleotide selection to catalysis Alignment of the trigger loop with the NTP and the precise positioning of a histidine side chain, 3.5 Å from the β-phosphate. The histidine promotes the flow of electrons during nucleophilic attack of the 3 - OH at the chain terminus and phosphoanhydride bond breakage. It serves as a proton donor for the pyrophosphate leaving group. It literally triggers phosphodiester bond formation. Strukturbiologie, Transcription 44

Nucleotide selection by alignment with the trigger loop, coupling recognition to catalysis The electronic transactions involved in trigger loop function require precise alignment of the interacting moieties. This is achieved for a correct NTP by formation of the trigger loop network. In the case of an incorrect NTP, for example a 2 -deoxy NTP, misalignment is profound. A double helix formed with a 2 -deoxy nucleotide is 2 Å narrower than that formed by a ribonucleotide. 45

Separation of RNA transcript from the template - 3D structure in the posttranslocation state -7-8 -9-10 Rudder Fork loop Lid Westover, K.D., et al. (2004) Structural basis of transcription: separation of RNA from DNA by RNA polymerase II. Science. Release of RNA transcript from DNA -RNA hybrid revealed in the structure of an RNA polymerase II transcribing complex. The upstream end of the DNA - RNA hybrid helix, 7-10 residues from the active center, is shown on the left, with distances between the DNA and RNA bases indicated. The entire DNA -RNA hybrid helix is shown on the right, along with protein loops involved in helix melting (rudder and lid) and stabilization (fork loop). Strukturbiologie, Transcription 46

Separation of RNA transcript from the template - 3D structure in the posttranslocation state -7-8 -9-10 Rudder Fork loop Lid Westover, K.D., et al. (2004) Structural basis of transcription: separation of RNA from DNA by RNA polymerase II. Science. Base pair 7 of the DNA- RNA hybrid in this structure appears normal the bases are coplanar, with a distance appropriate for hydrogen bonding between them. Base pairs 8, 9, and 10, however, show increasing deviations, and consequent splaying apart of the DNA and RNA strands. The strand separation is due to the intervention of three protein loops, termed fork loop 1, rudder, and lid. Strukturbiologie, Transcription 47

Separation of RNA transcript from the template - 3D structure in the posttranslocation state -7-8 -9-10 Rudder Fork loop Lid Westover, K.D., et al. (2004) Structural basis of transcription: separation of RNA from DNA by RNA polymerase II. Science. Rudder and lid lie between DNA and RNA. Rudder contacts DNA, Lid RNA. A Phe side chain of the lid serves as wedge to maintain separation of the strands. Fork loop contacts the sugar-phosphate backbone of the hybrid helix at base pairs 6 and 7, stabilizing the helix, preventing the DNA-RNA hybrid from unraveling further and inhibiting transcription. Strukturbiologie, Transcription 48

Transcription regulation: the role of Mediator Mediator is a key regulator of eukaryotic transcription, connecting activators and repressors bound to regulatory DNA elements with Pol II. Strukturbiologie, Transcription 49

Transcription regulation: the role of Mediator In the yeast Saccharomyces cerevisiae, Mediator comprises 25 subunits with a total mass of more than one megadalton and is organized into three modules, called head, middle/arm and tail. Architecture of the Mediator head module, nature 2011; x-ray structure of mediator head; 4.3 A Cryo-EM structure, 35 Å resolution, Asturias Lab 2002; Extension of the structure to atomic resolution will one day reveal the regulatory mechanism Strukturbiologie, Transcription 50

Transcription regulation: the role of Mediator In the yeast Saccharomyces cerevisiae, Mediator comprises 25 subunits with a total mass of more than one megadalton and is organized into three modules, called head, middle/arm and tail. Structure of the Mediator head module: Laurent Larivière, et al, Nature, 492, 448 451, (20 December 2012); 3.4 Å 51