German Edition: DOI: 10.1002/ange.201608066Gene TranscriptionInternational Edition: DOI: 10.1002/anie.201608066

The Structural Basis of Transcription: 10 Years After theNobel Prize in ChemistryMerle Hantsche and Patrick Cramer*

gene transcription · protein–nucleic acid complexes ·RNA polymerase · structural biology ·transcription regulation

1. Introduction

Ten years ago, in 2006, the Nobel Prize in Chemistry wasawarded to Roger Kornberg for “… studies of the molecularbasis of eukaryotic transcription”.[1] Kornberg and colleaguessolved the first structure of a eukaryotic RNA polymerase,the ten-subunit RNA polymerase II (Pol II) core enzymefrom the bakerQs yeast Saccharomyces cerevisiae.[2] Pol II isthe central enzyme that transcribes protein-coding genes andsynthesizes messenger RNA (mRNA), which in turn serves asa template to direct protein synthesis. With a molecularweight of around 500 000, the Pol II core structure was thelargest fully asymmetric protein complex solved by X-raycrystallography at the time.

The atomic details obtained from the yeast Pol II struc-ture[2] and a related structure of a bacterial RNA poly-merase,[3] determined by the laboratory of Seth Darst aroundthe same time, provided first insight into the mechanisms usedby these molecular machines, and were the starting point fora detailed structural characterization of gene transcription in

the years to follow (Figure 1). Thesedevelopments were possible due totechnological advances in structuralbiology. Whereas X-ray crystallogra-phy and biochemical probing were thestructural methods of choice for a longtime, cryo-electron microscopy (cryo-EM) recently developed to a stage

where it allows the study of large assemblies at near-atomicresolution.[4]

In this review, we summarize the progress made sincethese pioneering studies at the turn of the millennium. Inparticular, we discuss our current understanding of thestructure and mechanism of eukaryotic RNA polymerases.In eukaryotic cells, three multisubunit RNA polymerases,Pol I, Pol II, and Pol III, transcribe different classes of genesand produce various types of RNA. While Pol II synthesizesmessenger RNA (mRNA), Pol I and Pol III produce mainlyribosomal RNA (rRNA) and transfer RNA (tRNA), respec-tively. We focus on Pol II because it is the most studied ofthese enzymes. We describe structural advances in chrono-logical order for each of the three stages of the transcriptioncycle: initiation, elongation, and termination (Figure 2). Atthe end of each section, we mention open questions andfuture directions.

2. Transcription Initiation

RNA polymerase must first locate the promoter DNAsequence at the beginning of a gene, unwind the DNA duplex,and use the DNA template single strand to initiate RNAsynthesis.[5] Transcription initiation is a highly regulated

Transcription is the first step in the expression of genetic informationin all living cells. The regulation of transcription underlies cell differ-entiation, organism development, and the responses of living systemsto changes in the environment. During transcription, the enzyme RNApolymerase uses DNA as a template to synthesize a complementaryRNA copy from a gene. Herein, we summarize the progress in ourunderstanding of the structural basis of eukaryotic gene transcriptionthat has been made in the ten years since the Nobel Prize in Chemistrywas given to Roger Kornberg in 2006. The basis for transcriptioninitiation and RNA chain elongation is emerging, but the intricatemechanisms of transcription regulation remain to be elucidated. Thefield has also developed hybrid methods for structural biology thatcombine several techniques to determine the three-dimensionalarchitecture of large and transient macromolecular assemblies.

[*] M. Hantsche, Prof. P. CramerAbteilung ffr MolekularbiologieMax Planck Institut ffr biophysikalische ChemieAm Fassberg 11, 37077 Gçttingen (Germany)E-mail: pcramer@mpibpc.mpg.de

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process; it preferentially occurs at those genes that areexpressed in a certain cell type. Pol II initiation requires fivegeneral transcription factors that position Pol II on promoterDNA, assist Pol II in finding the transcription start site, andfacilitate DNA opening and initial RNA synthesis. In theclassical model,[6] transcription factor II D (TFIID) or itssubunit TATA-box binding protein (TBP) bind promoterDNA and TFIIB, which can then recruit the Pol II/TFIIFcomplex. Then TFIIE and TFIIH bind to the assembly toform a pre-initiation complex (PIC) on closed, double-stranded promoter DNA. Next, promoter DNA is unwoundin an ATP-dependent manner, resulting in a DNA “bubble”and the formation of an open complex (OC). RNA synthesisthen leads to an initially transcribing complex (ITC). Whenthe RNA grows to a critical length, Pol II escapes thepromoter, forms a stable elongation complex (EC), andexchanges general initiation factors for elongation factors

(Figure 2).[7] Initiation requires the complete 12-subunit formof Pol II, which includes the subcomplex Rpb4/Rpb7[8] thatwas lacking in the initial analysis of the Pol II structure.[2b]

2.1. DNA Promoter Recognition

Starting in the 1990s, several studies provided insight intohow promoter DNA is recognized by general transcriptionfactors.[5c] As a result, we learned how such factors bind toDNA and to each other to form a promoter assembly thatmarks the beginning of a gene. Eukaryotic promoters containvarious sequence elements that interact with components ofthe general transcription factors and are located around thetranscription start site (Figure 2).[9] The first identifiedeukaryotic promoter element was the TATA box, an AT-richsequence of eight base pairs,[10] which is specifically recog-

Patrick Cramer studied Chemistry at theUniversities of Stuttgart, Heidelberg, andBristol. He obtained his PhD in 1998 basedon structural studies of DNA complexes ofthe transcription factor NF-kappaB in thegroup of Christoph Mfller at the EMBL inGrenoble. After postdoctoral work in thegroup of Roger Kornberg at Stanford Uni-versity, where he solved the crystal structureof RNA polymerase II, he obtained one ofthe first tenure-track professorships in Ger-many in 2001, at the Gene Center of theLudwig-Maximilians-Universit-t (LMU) in

Munich. In 2004, he became a full professor at LMU and served asDirector of the Gene Center from 2004 to 2013. Since 2014, he has beenDirector at the Max-Planck-Institute for Biophysical Chemistry in Gçttin-gen.

Merle Hantsche received her diploma inbiochemistry from the University of Tfbin-gen. Since 2012, she has been a Ph.D.student in the group of Patrick Cramer,working on the structural characterization oftranscription initiation.

Figure 1. Selected atomic models of Pol II transcription complexes obtained through structural biology since the turn of the millennium. Thefollowing structures are shown in a previously defined “top view”:[2b] core Pol II[2b] and core Pol II elongation complex (EC; PDB ID: 1I6H),[2c]

complete Pol II[8a] and Pol II/TFIIS complex (PDB ID: 1PQV),[56a] complete Pol II/TFIIB complex (PDB ID: 3K1F),[21a] mammalian Pol II EC (PDB ID:5FLM),[26a] and core pre-initiation complex (PIC; PDB ID: 5FYW).[28]

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nized by TBP. Co-crystal structures revealed that TBPinduces DNA bending by almost 90degrees.[11] Adjacentbinding of TFIIA stabilizes the TBP/DNA complex.[12]

The pseudo-symmetric structure of TBP raised an im-portant question, namely how TBP binding could conferdirectionality on transcription, that is, how it ensures that thepolymerase moves in the right direction. Although this is stillnot fully understood, it appears that TBP has some preferencefor binding only in one orientation, and additionally, theadjacent factor TFIIB determines directionality. TFIIB bindsthe TBP/DNA complex such that it specifically contacts DNAelements on either side of the TATA box.[13] Since TFIIBbridges to Pol II, it can load Pol II in a unidirectional manner.

Biochemical and structural studies have generally beencarried out on TATA-box-containing promoters, although thismotif is only present at around 20% of eukaryotic promot-ers.[10] However, TBP is found bound to both TATA-contain-ing and TATA-less promoters in vivo.[10, 14] TBP is a subunit ofthe multiprotein complex TFIID, which contains 13–14 TBP-associated factors (TAFs).[15] TAFs contribute to promoterrecognition and mediate specific interactions with differentpromoter elements around the transcription start site.[16] Low-resolution EM studies revealed a horseshoe-like density thatadopted two different conformations.[17] Recently, a cryo-EMstructure of human TFIID bound to promoter DNA provideda more detailed view of TFIID–promoter interactions.[18]

TFIID forms two major contacts to promoter DNA, firstthrough TBP, which binds to the TATA element, and second,through TAF1, which binds to downstream promoter ele-ments. TFIID may thus act as a molecular ruler to positionTBP upstream of the transcription start site also at TATA-lesspromoters.

Taken together, eukaryotic promoters are composed ofDNA elements that are recognized by TFIID and TFIIB, withthe TFIID subunit TBP playing a central role. So far,recruitment of the general Pol II machinery has been studiedon only a few TATA-containing promoters, and alternativerecruitment pathways for TATA-less promoters are areas for

future studies. Finally, since promoter sequences are poorlyconserved, it is generally assumed that promoter recognitionalso involves indirect readout of other DNA characteristicssuch as bendability.

2.2. Opening and Loading of Promoter DNA for RNA ChainInitiation

Based on this progress in our understanding of promoterrecognition, the next step was to investigate how thepromoter assembly can recruit and position the polymeraseat the beginning of a gene. Since TFIIB is the central bridgebetween promoter DNA and the polymerase, the location ofTFIIB on the polymerase surface had to be determined. Earlystudies positioned the TFIIB amino-terminal domain onPol II, but this did not enable conclusive modeling of thepromoter assembly on Pol II.[19] Localization of the carbox-yterminal region of TFIIB using biochemical assays led toa topological model for the Pol II/promoter assembly com-plex and suggested that promoter DNA runs over thepolymerase active center cleft.[20] Crystal structures of Po-l II/TFIIB complexes confirmed and refined this model.[21]

These studies led to models of the closed and open complexesand revealed that around 30 nucleotides of DNA are requiredto connect the TATA element with the active center ofPol II,[21a] which explains the minimal distance between theTATA box and the transcription start site.[22] Further func-tions of TFIIB were revealed when the Pol II/TFIIB complexwas crystallized in the presence of a DNA scaffold witha short RNA.[23] Binding of TFIIB allosterically rearrangedthe catalytic site of Pol II, thereby stimulating RNA synthesis.

Co-crystallization of Pol II with other general initiationfactors was not successful. Instead, protein cross-linking andbiochemical probing were used to locate the general tran-scription factors TFIIE, TFIIF, and TFIIH on the Pol IIsurface.[24] Visualization of the topology of a complete pre-initiation complex was achieved by cryo-EM in 2013.[25] The

Figure 2. A schematic representation of the transcription cycle. Pol II binds the promoter of a gene close to the transcription start site (TSS) withthe help of initiation factors. During elongation, the nascent mRNA chain is extended. The polyadenylation (poly(A)) site marks the end of thegene where the mRNA is cleaved. Further downstream, Pol II is displaced from the DNA template and freed for a new round of transcription. ThemRNA is further processed through addition of a 5’ cap (black dot) and a poly(A) tail (An) at the 3’ end.

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architecture of the human complex was consistent with thesuggested models for the yeast complex derived frombiochemical studies.[24b,e,g] Subsequent cryo-EM structures ofhuman and yeast Pol II complexes underlined the highconservation between both systems.[26] The limited resolutionof these initiation complex densities, which ranged from 6 to13 c, still allowed docking of known structures of tran-scription factors into the densities.[11b, 12a,20b, 23, 27]

For mechanistic insight into Pol II initiation, higherresolution was required. This was achieved only very recently,when cryo-EM studies of the yeast[28] and human[29] systemsresulted in high-resolution information on several initiationassemblies. These studies showed that the general tran-scription factors TFIIB, TFIIE, and TFIIF are highly modular,with different structured domains being connected by flexiblelinkers. The cryo-EM structures revealed their inducedfolding upon assembly, and their intricate interactions witheach other, with Pol II, and with promoter DNA. From theseinsights, a modified view of promoter recognition emerges.An intricate, transient network of protein–protein andprotein–DNA interactions, which mainly responds to globalfeatures of the DNA, apparently underlies the positioning ofPol II at promoters.

These recent studies also provided insight into DNAunwinding. Comparison of complexes containing eitherclosed or open promoter DNA showed movements mainlyin TFIIE, thus indicating a role for TFIIE in promoteropening or the stabilization of open DNA. TFIIE also recruitsTFIIH,[30] which contains a DNA translocase that functions inpromoter opening.[5b,c,31] How TFIIH achieves promoterDNA unwinding and loading into the Pol II active-centercleft has been studied biochemically.[24a,e, 32] Structural infor-mation supports the functional findings.[21a, 25,26b, 29] TFIIHbinds DNA downstream and translocates along DNA awayfrom Pol II. Since both the promoter and TFIIH are held in

the pre-initiation complex, this translocase action createstorsional stress on the DNA, thereby facilitating its unwindingand pushing downstream DNA into the active-center cleft.The emerging DNA template strand is threaded into theactive center, where it is bound by an element of TFIIB, the“B-reader”, which functions in recognizing the transcriptionstart site.[21a] Although there is no question about the functionof TFIIH in promoter opening, DNA opening can also occurin the absence of TFIIH in vitro,[28] which is consistent withearly functional studies.[33]

Taken together, structural advances over the last fewyears have provided a detailed picture of the intricatearchitecture of the Pol II initiation complex, explaining manyfunctions of the general transcription factors. Future studieswill have to expand these studies to TFIID and TFIIH, whichare at present not resolved to high resolution (Figure 3).

2.3. Regulation of Initiation

Promoter elements are located in the vicinity of thetranscription start site. There are, however, multiple addi-tional DNA sequence elements outside of the promoter thatbind regulatory proteins. These regulatory elements arelocated near the promoter, but in multicellular organismscan also be located at some distance from the promoter, andare then called enhancers. In order to convey a signal froma regulatory-DNA-bound transcription factor to the poly-merase, a bridging co-activator is required.

Mediator is a central co-activator needed for transcriptionof most protein-coding genes.[34] Though discovered over twodecades ago,[35] the molecular mechanisms Mediator uses arenot well understood because structural studies have beenimpeded by the size, flexibility, and modularity of this largecomplex. In yeast, Mediator has a molecular weight of

Figure 3. A composite topological model of the Pol II initiation complex. Cryo-EM densities of TFIID (EMD-3305),[18] TFIIH (EMD-3307),[18] andMediator head and middle modules (EMD-2786)[26c] are superposed on the structure of Pol II, together with TFIIA, TFIIB, TBP, TFIIF, TFIIE, andpromoter DNA (PDB ID: 5FZ5).[28]

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1.4 MDa and is composed of 25 proteins, and metazoanMediator contains several additional subunits. Based on earlystudies, the Mediator structure was divided into four modules:the head, middle, tail, and kinase modules.[36] Many EMstudies of Mediator were reported, but at low resolution,leaving many uncertainties.[37] The Mediator head moduleinteracts with the general Pol II initiation machinery, the tailbinds gene-specific transcription factors, and the kinasemodule can modify the flexible C-terminal domain (CTD)of the large Pol II subunit. The CTD is a unique Pol IIelement that serves as a platform for recruiting proteins forco-transcriptional processes, and it changes its phosphoryla-tion pattern during the transcription cycle.[38]

Recent advances in the structural biology of Mediatorhave elucidated its interaction with the Pol II initiationmachinery.[39] Co-expression enabled the preparation of the7-subunit Mediator head module,[40] the 7-subunit middlemodule,[41] and later of the core Mediator comprising the headand middle modules and Med14, a subunit that forms anarchitectural backbone.[26c,42] The crystal structure of the headmodule was solved,[40b,43] based on structures of subcomplex-es.[39a] The structure of a core Mediator/initiation complex wasdetermined by cryo-EM and showed that the Mediator headmodule binds on one side of Pol II, where it can stabilizeTFIIB and the polymerase stalk, which is involved ininitiation.[26c] Mediator and TFIID apparently bind to oppo-site sides of Pol II, with TFIIH in between (Figure 3).[18] Suchembedding of Mediator in the initiation assembly explainshow it stabilizes the PIC. The tail module communicates withtranscription factors.[44] Activator binding triggers conforma-tional changes in Mediator, but how these are transmitted andhow they influence interaction with the initiation complex isunknown.[45]

3. Elongation of the RNA Chain

3.1. Addition of RNA Nucleotides

During RNA chain elongation, the polymerase repeatsthe so-called nucleotide addition cycle. In each cycle, Pol IIpresents the template DNA base, selects and binds a comple-mentary RNA nucleoside triphosphate (NTP) substrate,catalyzes phosphodiester bond formation to add a nucleotideto the growing RNA chain, and translocates to the nexttemplate position, thereby freeing the NTP-binding site. Awealth of crystal structures of Pol II with various DNA–RNAscaffolds have elucidated the nucleotide addition cycle andmany other aspects of elongation (Figure 4).[46] Studies in thebacterial system have contributed substantially to our under-standing of elongation, because all polymerases share a con-served catalytic center and transcription mechanism.[2a,b, 3, 47]

In the first step of the nucleotide addition cycle, thecorrect NTP is selected by the Pol II active center ina sampling process.[48] The NTP binds transiently to an openand catalytically inactive pre-insertion conformation. Uponbase-pairing of the NTP with the template base, the active sitecloses through folding of the trigger loop and the NTP ismoved to the insertion site and positioned for catalysis(Figure 4b). This two-step mechanism of NTP selection is alsoused by the bacterial RNA polymerase.[49] The mechanism forcatalytic nucleotide addition was proposed based on struc-tural studies of DNA polymerases.[50] The mechanism involvestwo magnesium ions: one (metal A) persistently bound in theactive center, and the other (metal B) mobile.[2b] Both metalions are coordinated by conserved negatively charged aminoacid residues. Metal A binds the RNA 3’ end and metal Bpositions the NTP moiety (Figure 4b). Catalysis occurs

Figure 4. Polymerase active center and nucleic acid interactions. a) A central slice through the Pol II elongation complex, indicating the path ofthe DNA (template strand, blue; non-template strand, cyan) and newly synthesized RNA (red). Important elements of the active center arehighlighted: active center (magenta), bridge helix (green), and trigger loop (yellow; PDB ID: 4A3F).[48e] b) A detailed view of the nucleic acids andthe active center. The NTP substrate is a non-hydrolysable nucleoside triphosphate (AMPCPP).[48e] Metal A is permanently bound and coordinatedby three conserved aspartate residues (D481, D483, and D485). For catalysis, a second magnesium ion (metal B) is recruited, which was modeledaccording to the bacterial EC (PDB ID: 2O5J).[49a]

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through a nucleophilic substitution (SN2) mechanism, inwhich the RNA 3’-OH group acts as the nucleophile andattacks the NTP a-phosphate. Release of diphosphate mayinduce opening of the active center.

After nucleotide addition, Pol II has to translocate by onestep along the DNA, to free the NTP-binding site that isoccupied by the newly added RNA 3’ nucleotide. Structuralstudies of Pol II bound by the mushroom toxin a-amanitinhelped to reveal the translocation mechanism.[51] Amanitinbinds beneath two Pol II elements in the active center, thetrigger loop and bridge helix, which form a Brownian ratchetthat rapidly switches between pre- and a post-translocationstates (Figure 4a). NTP binding to the active center acts likethe pawl of the ratchet. Only after addition of the nucleotideto the growing RNA, does the oscillations resume, moving thenascent hybrid base pair out of and the next template baseinto the active site. Directionality is achieved since NTPs canonly bind in the post-translocation state of the ratchet. Thetoxin a-amanitin traps the ratchet in an intermediary state.[51c]

From these studies, the basic DNA template-directed RNAsynthesis mechanism was derived.

3.2. Dealing with Errors and Obstacles

Transcription elongation is prone to errors and often facesobstacles. Structural studies have elucidated how errors canbe prevented and corrected. Transcription fidelity is to a largeextent achieved through selection of the correct, cognateNTP, but is further improved through a “proofreading”mechanism.[46b, 48c,52] After nucleotide misincorporation, themismatched nascent base pair generally distorts the activesite, causing the polymerase to pause, which can involvefraying of the 3’ terminal, mismatched nucleotide.[48d] Pol IIcan then backtrack by one step to move the mismatchednucleotide into a proofreading site. The intrinsic endonu-clease activity of Pol II is then stimulated, resulting incleavage of a RNA dinucleotide that contains the mis-match.[53] Afterwards Pol II can resume transcription.

Structural studies have also provided insight into how thepolymerase overcomes obstacles. During elongation, Pol IImay run over DNA sequences that destabilize the DNA–RNA hybrid (such as AT-rich sequences). This often causesPol II to reverse direction and undergo backtracking, whichleads to arrest if it is extensive. During backtracking, the RNA3’ end is dislodged from the active center and inserted intoa pore beneath the active site.[54] Binding of backtrackedRNA to a site in the pore apparently leads to polymerasearrest. Pol II cannot escape from arrest by itself. To restartelongation, it requires the help of an external factor, TFIIS,which induces RNA cleavage to form a new 3’ end adjacent tothe Pol II active site.[55] Structural studies of Pol II in complexwith TFIIS have revealed the mechanisms of RNA cleavageand Pol II reactivation.[48b,54, 56] TFIIS binds to the Pol IIsurface near the pore and reaches the active site with a thinhairpin. The conserved zinc-ribbon domain of TFIIS fulfillstwo tasks; it releases the backtracked RNA from the back-track site, and it induces RNA cleavage by complementing thePol II active site with additional catalytic residues. These

studies showed that Pol II has a single, tunable active site thatcan switch between polymerization and cleavage modes.[56a]

When Pol II encounters a DNA lesion, different outcomesare possible. Whereas bulky lesions trigger Pol II pausing,small lesions can be bypassed and may lead to transcriptionalmutagenesis. 8-Oxoguanine, a base modification that resultsfrom oxidative damage, can lead to misincorporation becausean uncommon syn-conformation of the base in the activecenter can lead to a Hoogsteen base pair with adenine.[57]

Pol II elongation rates are significantly reduced when tran-scribing through genes with oxidized intermediates of 5-methylcytosine, which suggests that Pol II can specificallysense epigenetic marks.[58] Bulky DNA lesions, such asultraviolet-light-induced cyclobutane pyrimidine dimers(CPDs) or intrastrand crosslinks caused by the anticancerdrug cisplatin, generally lead to stable stalling of Pol II andsubsequent initiation of transcription-coupled repair,[59] al-though mechanisms differ.[60]

The elongation complex is very stable mainly because ofthe stability of the DNA-RNA hybrid in the active center.[61]

Additionally, elongation factors repress pausing and enhancePol II processivity, that is, the ability of Pol II to remainassociated with the template DNA until transcription isterminated. Spt5 is a highly conserved and ubiquitouselongation factor.[62] Structural studies of this factor[63] andits complex with polymerase[26a, 64] have shown that a con-served domain of Spt5 spans the active center cleft, lockingnucleic acids in the cleft and thus preventing elongationcomplex dissociation. Elongation factors also function in co-transcriptional processes, such as 5’ RNA capping, splicing,and chromatin remodeling.[62] The molecular basis andcoordination of these processes represent important areasfor future studies.

3.3. Termination of Elongation

At the end of the transcription cycle, Pol II must releasethe RNA transcript and dissociate from the DNA template(Figure 2). Correct transcription termination is important toprevent transcription interference at downstream genes andto enable recycling of Pol II.[47c,65] When the polymerase runsover the polyadenylation (poly(A)) signal, which marks theend of a gene, the nascent RNA chain is cleaved and a poly(A)tail is added to the 3’ end of the RNA. Pol II howeverterminates further downstream at multiple positions. Recentwork has shown that human genes contain on average fourtermination sites within a window of several thousand basepairs downstream of the poly(A) site.[66]

Two different models for Pol II termination have beensuggested. In the allosteric model, the binding of RNA 3’-processing factors induces structural rearrangements in thePol II elongation complex that elicit termination.[67] In thetorpedo model, a nuclease degrades the newly synthesizedRNA after cleavage, catches up with the elongation complex,and thereby dislodges Pol II from DNA.[68] The protein Rat1in yeast (Xrn2 in humans) is the torpedo nuclease.[69] There isalso evidence for a unified mechanism that includes aspectsfrom both models.[70]

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The structural mechanism of transcription terminationstill remains enigmatic. There is evidence that the Pol IIelongation complex slows down and changes its factorcomposition and its phosphorylation state when Pol II runsover the poly(A) site,[70b, 71] but the nature of the pre-termination complex is currently under discussion.[72] Thetransient nature of the pre-termination complex has thus farprevented its structural analysis, but it is likely that termi-nation involves repositioning of Spt5 and widening of thePol II cleft to facilitate release of the nucleic acids.

4. Polymerase Conservation and Gene Class Specif-icity

The eukaryotic genome is transcribed not only by Pol II,but also by Pol I and Pol III, which synthesize mainlyribosomal RNA (rRNA) and transfer RNA (tRNA), respec-tively. Whereas Pol II makes thousands of different mRNAs,most RNA in the cell is made by Pol I and Pol III, whichsynthesize around 80 % of the total RNA transcripts inyeast.[73] The three polymerases share a 10-subunit core anda conserved two-subunit stalk (Figure 5).[74] Recent high-resolution structures of yeast Pol I[75] and Pol III[76] haverevealed that the polymerases also share the same active siteand apparently apply the same catalytic mechanism for thenucleotide addition cycle. However, the polymerases differ intheir molecular size and number of subunits. Compared toPol II, Pol I and Pol III contain additional subunits on theirperiphery that resemble domains of the general transcriptionfactors TFIIE and TFIIF, and the cleavage-stimulatory factorTFIIS (Figure 5).[77]

The differences between the polymerase surfaces reflectgene-class-specific requirements for transcribing and regulat-ing different RNA transcripts. Like Pol II, Pol I and Pol IIIrely on a specific set of initiation factors that control therecruitment and positioning of the polymerases to theirdistinct promoter types.[78] TBP and a TFIIB-like factor are

apparently universally required.[79] For Pol II, initiation re-quires the ATP-consuming factor TFIIH, whereas the twoother polymerases can open DNA without ATP consumption.Therefore, Pol I and Pol III contain subdomains as integralparts that are important for transcription initiation andprocessivity. In the Pol II system, these domains formseparate factors, probably due to an extended need forregulatory control. The structural characterization of Pol Iand Pol III initiation complexes will be an important next stepin understanding promoter specificity of the three poly-merases, and eventually their distinct modes of regulation.

5. Conclusions

At the turn of the millennium, when we first saw thestructure of Pol II, we were fascinated by its beauty andintricacy. However, the polymerase structure marked only thebeginning of a long and winding road towards an under-standing of the transcription mechanism, which is a fascinatingproduct of molecular evolution that involves transient anddynamic interplay of the polymerase with nucleic acids anddozens of protein factors. By now, the molecular mechanismsof transcription initiation and elongation are emerging, butthere are still many open questions. Also, we still understandvery little about the mechanisms that underlie transcriptionregulation and transcription in the context of chromatin, thenatural DNA template. The black box of transcription thuscontinues to be opened, step by step, as more structures ofever larger polymerase complexes are resolved. The pace atwhich we move is ever increasing as a result of recentadvances in protein complex preparation and cryo-EM. Weare therefore hopeful that in another ten years, significantadditional insight into the structural basis of transcription willbe available. Eventually we hope to reach a mechanisticunderstanding of how genes are switched on and off.

Figure 5. Conservation between eukaryotic polymerases. Structures of Pol I (PDB ID: 4C2M),[75a] Pol II (PDB ID: 1WCM),[80] and Pol III (PDB ID:5FJ9)[76] are shown in “front view”.[2b] Pol II is shown together with a part of TFIIE (PDB ID: 5FYW),[28] the dimerization domain of TFIIF (PDB ID:5FYW),[28] and TFIIS (PDB ID: 3PO3).[54] Subunits of Pol I and Pol III that are structurally and functionally similar to TFIIE, TFIIF, and TFIIS, arecolored according to Figure 1. The C-terminal part of A49 (PDB ID: 3NFH)[77b] and the subunit C34 show some homology with TFIIE (pink), thesubcomplex A49/34.5 and C37/53 are homologous to the dimerization domain of TFIIF (purple), and the subunits A12.2 and C11 are homologsof TFIIS (orange). The Pol II stalk, cleft, and clamp are labeled.

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Acknowledgements

M.H. was supported by the Deutsche Forschungsgemein-schaft (GRK1721). P.C. was supported by the DeutscheForschungsgemeinschaft (SFB860, SPP1935), the AdvancedGrant “TRANSREGULON” of the European ResearchCouncil, and the Volkswagen Foundation.

How to cite: Angew. Chem. Int. Ed. 2016, 55, 15972–15981Angew. Chem. 2016, 128, 16204–16214

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Received: August 19, 2016Published online: November 11, 2016

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15981Angew. Chem. Int. Ed. 2016, 55, 15972 – 15981 T 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.angewandte.org