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Kulbachinskiy and Katsuhiko S. MurakamiCarlos Fernndez-Tornero, AndreyMolodtsov, Danil Pupov, Daria Esyunina,Ritwika S. Basu, Brittany A. Warner, Vadimby Bacterial RNA Polymerase HoloenzymeStructural Basis of Transcription InitiationGene Regulation:

doi: 10.1074/jbc.M114.584037 originally published online June 27, 20142014, 289:24549-24559.J. Biol. Chem.

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Structural Basis of Transcription Initiation by Bacterial RNAPolymeraseHoloenzyme*SReceivedfor publication, May 22,2014, andin revised form, June 23,2014 Published,JBC Papers in Press,June 27, 2014, DOI 10.1074/jbc.M114.584037

Ritwika S. Basu, BrittanyA.Warner1, VadimMolodtsov, Danil Pupov, Daria Esyunina,

Carlos Fernndez-Tornero

, Andrey Kulbachinskiy

, and Katsuhiko S.Murakami2

From theDepartment of Biochemistry and Molecular Biology, The Center for RNA Molecular Biology, The Pennsylvania StateUniversity, University Park, Pennsylvania 16802, Centro de Investigaciones Biolgicas, Consejo Superior de InvestigacionesCientficas, Ramiro de Maeztu 9, 28040 Madrid, Spain, and theLaboratory of Molecular Genetics of Microorganisms, Institute ofMolecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia

Background:Cellular RNA polymerases start transcription byde novoRNA priming.Results: Structures and biochemical studies of initially transcribingcomplexes elucidate the de novo transcription initiation andearly stage of RNA transcription.Conclusion:5-end of RNA in the transcribing complex starts ejection from core enzyme.Significance:Insights from this study can be applicable to all cellular RNA polymerases.

The bacterial RNA polymerase (RNAP) holoenzyme contain-

ing factor initiates transcription at specific promoter sites by

de novo RNA priming, the first step of RNA synthesis where

RNAP accepts two initiating ribonucleoside triphosphates

(iNTPs) and performs the first phosphodiester bond formation.

We present the structure of de novo transcription initiation

complex that reveals unique contacts of the iNTPs bound at the

transcription start site with the template DNA and also with

RNAP and demonstrate the importance of these contacts for

transcription initiation. To get further insight into the mecha-

nism of RNA priming, we determined the structure of initially

transcribing complex of RNAP holoenzyme with 6-mer RNA,

obtained byin crystallotranscription approach. The structure

highlights RNAP-RNA contacts that stabilize the short RNAtranscript in the active site and demonstrates that the RNA

5-end displaces region 3.2 from its position near the active

site, which likely plays a key role in ejection during the initia-

tion-to-elongationtransition. Given the structuralconservation

of theRNAP activesite,the mechanismofde novo RNA priming

appears to be conserved in all cellular RNAPs.

During successive steps of transcription initiation, RNAP3

must employ different mechanisms of NTPloading andaccom-modate the growing RNA product within the RNA channel

until entering the stable transcription elongation phase.All cel-lular and most bacteriophage RNAPs initiate transcription denovoby loading two iNTPs opposite the first and second tem-plate DNA bases at the transcription start site to synthesize thefirst phosphodiester bond of RNA. Loading the first iNTP,which will become the 5-end of RNA, at theisite is a uniqueprocess forde novotranscription by RNAP because this site inthe transcription elongation complex usually accommodatesthe RNA 3-end, whose binding is stabilized by the preceding8 base pairs of the DNA/RNA hybrid (1). The bindingaffinityof the first iNTP duringde novotranscription is substantiallylowerthanthatofthesecondiNTPbindingatthei1site(2,3).This allows the direct sensing of NTP concentrations by RNAP

to become a basis of regulating transcription initiation at ribo-somal RNA promoters (4) and as well at the pyrimidine biosyn-thesis genes (5) in bacteria. High-resolution crystal structuresof the de novo transcription initiation complex containingRNAP, DNA, and the first and second iNTPs bound at theactive site have been determined for bacteriophage T7 (6) andN4 (7) RNAPs, and these structures revealed unique interac-tions between the first iNTP and RNAP/DNA. However, due tostructural differences between different classes of RNAPs,insights from the bacteriophage RNAP structures cannot bedirectly transferred to cellular RNAPs. Previously publishedcrystalstructureoftheThermus thermophilus RNAP transcrip-

tion initiation complex contains a GpA dinucleotide primercomplementary to the template DNA positions1 and1 butlacking the 5-triphosphate group (8). Recently, a structure ofthe T. thermophilusRNAP initiating complex containing twoiNTPshasbeenreported(9).However,therolesoftheobservedRNAP-iNTP contacts in transcription initiation were nottested experimentally; in addition, the structure contained asuboptimal template strand sequence around the transcriptionstart site (see below), suggesting that it might miss some impor-tant contacts with the iNTPs.

Following the de novo incorporation step, RNAP goesthrough several cycles of NTP addition before entering tran-scription elongation, during which a highly stable and proces-

* Thisworkwassupported,inwholeorinpart,byNationalInstitutesofHealthGrant GM087350-A1 (to K. S. M.). This work was also supported in part bythe Spanish government through Grant BFU2010-16336 (to C. F.-T.). Thiswork was also supported by the Russian Foundation for Basic Research(Grants 14-04-01696 and 14-04-31994) and the Russian Academy of Sci-ences Presidium Program in Molecular and Cellular Biology (to A. K.).

The atomic coordinates and structure factors (codes 4Q5S and4Q4Z) have beendepositedin theProtein Data Bank (http://wwpdb.org/).

S This article containssupplemental Movie S1.1 Present address: Janssen Research and Development, L.L.C., Spring House,

PA 19477.2 To whom correspondence should be addressed: Dept. of Biochemistry and

Molecular Biology, The Pennsylvania State University, University Park, PA16802. Tel.: 814-865-2758; E-mail: [email protected].

3 The abbreviations used are: RNAP, RNA polymerase; iNTP, initiating ribonu-cleoside triphosphate; Pol II, RNA polymerase II.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 35, pp. 245 49 24559, August 29, 2014 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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sive RNAP complex performs synthesis of thousands of bases ofRNA. In contrast, the initially transcribing complexes of RNAPholoenzyme containing short RNAs, usually ranging 212nucleotides in length, are unstable resulting in abortive initia-tion (10, 11). Multiple events during initial transcription,including release from the core enzyme, DNA scrunching,and promoter escape, have been proposed (1214); however,

molecular basis of the initial transcription by bacterial RNAP islacking due to the limited stability of these complexes and thedifficulty of capturing a homogeneous initiating complex con-taining short nascent RNA.

Here, we report a crystal structure of the de novotranscrip-tion initiation complex ofT. thermophilusRNAP, containingtwo iNTPs loaded in the active site. The structure and alsostructure-based biochemical assays reveal key interactionsbetween the RNAP holoenzyme, DNA, and iNTPs that are crit-ical forde novotranscription initiation. Furthermore, we pre-pared a homogeneous initially transcribing complex with a6-mer RNA using in crystallo transcription approach andsolved its crystal structure, which provides insights into the

binding of short RNA-DNA hybrid in the active site and in theprocess ofrelease triggered by the nascent RNA, as the ini-tially transcribing complex begins transition into the elonga-tion phase of transcription.

EXPERIMENTAL PROCEDURES

Preparation and Crystallizationof the T. thermophilus RNAP

Promoter DNA Complex, the de Novo Transcription Initiation

Complex, and the Transcription Complex Containing 6-Mer

RNAT. thermophilus HB8 cells were cultured by using a 300-liter BioService fermentor at the Penn State fermentation facil-ity. EndogenousT. thermophilusRNAP core enzyme was puri-fied as follows: 100 g of cell paste was suspended in 300 ml oflysisbuffer(40mM Tris-HCl,pH8,at4 C,100mM NaCl,10mMEDTA, 10 mM2-mercaptoethanol, 1 mMbenzamidine, 1 mMPMSF, 0.5 g/ml leupeptin, and 0.1 g/ml pepstatin), and cellswere lysed by Emulsiflex C3 homogenizer (Avestin, Inc.) at20,000 psi. After 30 min, benzamidine and PMSF (1 mM) wereadded to the lysate. The lysate was then clarified by centrifuga-tion, and glycerol was added to the supernatant to a concentra-tion of 5%. RNAP in the soluble fraction was precipitated byadding 10% polyethyleneimine (Polymin-P) solution (0.5%),and the pellet was recovered by centrifugation. The pellet wasthen resuspended and washed with 200 ml of wash buffer (40mMTris-HCl, pH 8, at 4 C, 200 mMNaCl, 1 mMEDTA, 10 mM

2-mercaptoethanol, 5% glycerol, 1 mMbenzamidine, and 1 mMPMSF) and again recovered by centrifugation. This wash stepwas repeated with an additional 200 ml of wash buffer. Finally,RNAP was recovered by resuspending the pellet in 100 ml ofextraction buffer (40 mMTris-HCl, pH 8, at 4 C, 1 M NaCl, 1mMEDTA, 10 mM2-mercaptoethanol, 5% glycerol, 1 mMben-zamidine, and 1 mMPMSF) and centrifuging for 15 min at 4 Cand17,000rpm.Thisextractionstepwasrepeatedwithanother100 ml of extraction buffer, and the supernatants from bothextractions were combined (total, 200 ml). Ammonium sulfatepowder was then gradually added to 45% concentration to pre-cipitate RNAP followed by centrifugation. The pellet was sus-pended in TGED buffer (10 mMTris-HCl, pH 8 at 4 C, 0.1 mM

EDTA, 5% glycerol, and 1 mMDTT), and RNAP core enzymewas purified by heparin, ResourceQ, and SP Sepharose columnchromatography (GE Healthcare).

T. thermophilusA was expressed in BL21(DE3) cells trans-formed with a pET21a plasmid containingT. thermophilus sigAgene. Cells were grown in 1.5 L LB containing 100g/ml ampi-cillin for 6 h, induced with 1 mMisopropyl 1-thio--D-galacto-

pyranoside, and grown for additional 3 h at 37 C. After cellswere lysed by sonication,Awaspurifiedfromthelysatebyheattreatment in a 65 C water bath for 30 min. The suspension wascentrifuged at 16,000 rpm to remove the white precipitate. A

was then purified from the supernatant by ResourceQ column(GE Healthcare)chromatography.Awasstoredin50mM Tris-HCl, pH 7.7, 400 mMNaCl, and 15% glycerol at 20 C.

T. thermophilusRNAP holoenzyme was prepared by adding3-fold molar excess ofA to core RNAP and then purified bySuperdex200 column chromatography. The RNAP and pro-moter DNA complex was prepared by mixing 18 M(24 l)T. thermophilusholoenzyme (in 20 mMTris-HCl, pH 7.7, 100mM NaCl, and 1% glycerol) and 1 mM (0.65 l) of the DNA

scaffold (see Fig. 1A) and incubated for 30 min at 22 C. Crystalswere obtained by using hanging drop vapor diffusion by mixingequal volume of RNAP-DNA complex solution and crystalliza-tion solution (100 mMTris-HCl, pH 8.7, 200 mMKCl, 50 mMMgCl2, 10 mMSpermine tetra-HCl, and 10% PEG 4000) andincubating at 22 C over the same crystallization solution. Thecrystals were cryoprotected by soaking in same constituents asthe crystallization solution with stepwise increments ofPEG4000 and (2R,3R)-()-2,3-butanediol (Sigma-Aldrich) tofinal concentrations of 25 and 15%, respectively. The final cryo-protectant solution was also used for soaking NTPs at roomtemperature to prepare the de novo transcription initiationcomplex(5mM ATP;and5mM CMPCPPfromJena Bioscience;soaking time, 30 min) and the holoenzyme transcription com-plexwith6-merRNA(5mM each of ATP, CTP, and UTP; soak-ing time, 5 h).

X-ray Data Collections and Structure DeterminationThecrystals belong to the C-centered monoclinic space group(Table 1) containing oneT. thermophilusRNAP transcriptioncomplex per asymmetric unit. The data set was collected at theMacromolecular Diffraction at Cornell High Energy Synchro-tron Source (MacCHESS) F1 beamline (Cornell University,Ithaca, NY), and the data were processedby HKL2000 (15). TheT. thermophilusRNAP-promoter DNA complex (8) was usedas a search model for the molecular replacement (16). Rigid

body refinements were performed, and further adjustments tothe model were performed manually. The resulting modelphases allowed positioning of iNTPs in the de novotranscrip-tion initiation complex and RNA in the initially transcribingcomplex in their electron-density maps. Positional refinementand reference model restraints was performed by the programPhenix (17).

In Vitro TranscriptionThe wild-type Escherichia coliRNAP core enzyme and holoenzymes containing70 were pre-pared as described (18). Mutations in the E. coli rpoBgene ofthe RNAP subunit were obtained by site-directed mutagene-sisof plasmid pIA545 andrecloned into plasmid pIA679 encod-ing all four core RNAP subunits with a His6tag in the N termi-

Bacterial RNAPolymerasedeNovo Transcription

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nus of the subunit (both plasmids kindly provided by I.Artsimovitch). Mutant RNAPs were expressed in E. coliBL21(DE3) and purified using Polymin-P precipitation, hepa-rin, and nickel-nitrilotriacetic acid affinity chromatography,and MonoQ column ion exchange chromatography (19). TheE. coli RNAP holoenzyme containing thesubunit with region3.2 deletion was prepared as described (20).

In vitrotranscription ofE. coliRNAP containing wild-typeand mutant subunits was carried out as described (20).Apparent K

mvalues for iNTPs were measured as described

(20). RNA products were separatedby 15% (for full-length RNA

transcription), 23% or 30% (for abortive RNAs) PAGE, followedby phosphorimaging.Analysis of DNA Sequences around Transcription Start Site of

Human Pol II PromotersHuman RNAP II (Pol II) promotersequences (10,000 of 25,976 in database) were obtained fromThe Eukaryotic Promoter Database (21) and analyzed for theirsequence conservations. Graphical representation was pre-pared using WebLogo (22).

RESULTS

Design of the X-ray Crystallographic Experiment to Deter-

mine the Structure of the de novo Transcription Initiation

ComplexThe T. thermophilusRNAP holoenzyme-promoter

DNA complex crystals were obtained by modifying a methoddescribed previously (8), using a DNA scaffold containing the10 and consensus discriminator elements (23) in thenontem-plate strand (Fig. 1A). To obtain the crystals of the de novotranscription initiation complex, the holoenzyme-promoterDNA crystals were soaked with the first two iNTPs that basepair with the 1 and 2 bases of template DNA. To maintainthe transcription bubble during crystallization of the holoen-zyme-promoter DNA complex, previous studies used a non-complementary template strand sequence upstream of thetranscription start site (8, 9). Our previous structural analysis ofthe N4 phage RNAPde novotranscription complex (7) identi-fied that the template DNA nucleotide at the 1 position par-

ticipatesin binding thefirst iNTP by a base stacking interaction.Therefore, in this study, we changed the template DNAsequence at the 1 position to a guanine base (the template-strand sequence 3-GTGA-5; the transcription start site isunderlined), thus mimicking the natural sequence of mostbacterial promoters recognized by the primaryfactor (24).By incubating the crystals with ATP and a nonhydrolyzableCTP analog, CMPCPP (cytidine-5-[(,)methyleno]tri-phosphate), we obtained thede novotranscription initiationcomplex structure with the two iNTPs bound at the activesite, but without phosphodiester bond formation. The struc-

ture was solved by molecular replacement at 2.9 resolu-tion. The structure showed strong unbiased FoF

celectron

densities corresponding to the first and second iNTPs (Fig.2A,supplemental Movie S1). The overall structure of RNAPwas identical with that of the search model (Protein DataBank code 4G7H) (8). However, important differencesincluded conformational changes in the trigger loop of thesubunit (residues12331255) and the amino acid side chainsof both the and subunits around the active site (Figs. 2,Band C).

The First iNTP Binding Site in the de Novo Transcription

Initiation ComplexBinding of the first and second iNTPs

involves interactions with residues around the active site thatare unique tode novotranscription and other interactions thatare common to RNA elongation. The first iNTP occupies the isiteinthe de novo transcription initiationcomplex, which over-laps with the RNA 3-end binding site in the elongation com-plex (Fig. 2C) (1). The de novotranscription-specific interac-tions between RNAP and iNTPs appear to concentrate on thetriphosphate of the first iNTP, which is ATP in our crystalstructure, with multiple salt bridges with the subunit resi-dues, including 1) Gln-567andHis-999 (interact with a non-bridging oxygen of-phosphate; correspond to E. coliRNAPresidues Gln-688 and His-1237); and 2) Lys-838 (interactswith a nonbridging oxygen of -phosphate; corresponds to

TABLE1

Datacollection and refinement statisticsData sets were collected at MacCHESS-F1 (Ithaca, NY).

Complex De novoa ITCb

PDB code 4Q4Z 4Q5SData collection

Space group C2 C2Cell dimensions a 184.49,b 102.16, andc 294.72 ; 98.96 a 187.09,b 102.08, andc 297.26 ; 98.14Resolution () 502.85 503.00

Total reflections 260,182 273,453Unique reflections 111,819 107,824Redundancy 2.3 (1.4)c 2.5 (1.9)c

Completeness (%) 88.3 (67.7)c 94.4 (78.8)c

I/ 5.4 (1.4)c 6.6 (1.2)c

Rsym 0.144 (0.508)c 0.124 (0.719)c

RefinementResolution () 502.90 503.00

Rwork 0.256 0.269Rfree 0.275 0.294r.m.s.d.d

Bond length 0.005 0.002 Bond angles 0.938 0.61

a Thede novotranscription complex of RNAP holoenzyme.b The initially transcribing complex of RNAP holoenzyme containing a 6-mer RNA.c Highest resolution shells are shown in parentheses.d r.m.s.d., root mean square deviation.

BacterialRNAPolymerase deNovo Transcription


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E. coli residue Lys-1065) (Fig. 2, Band E). Electron densitymap for the Lys-846 side chain (E. coliresidue Lys-1073) isnot well defined (data not shown),but it may be positioned neara nonbridging oxygen of-phosphate for making an additionalsalt bridge. Arg-704 of the subunit (Arg-704;E. coliresi-dueArg-425)formssaltbridgeswith2 -OHand3-OHoftheATP and may therefore participate in discrimination between

NTP and dNTP. All residues interacting with the ATP triphos-phate are absolutely conserved in all cellular RNAPs (Fig. 2E),suggesting a universal mechanism for accommodating the firstiNTP at the enzyme active site to establish de novotranscription.

The structure is consistent with previous biochemical stud-ies showing that residues Lys-1065, Lys-1073, and His-

FIGURE 1. Structure of the transcription initiation complex.A, schematic of thede novotranscription initiation complex (top) and the initially transcribingcomplex (bottom) obtained from the RNAP-promoter complex. Sequence of the nucleic acid scaffold used for holoenzyme-promoter DNA complex crystalli-zation is shown in themiddle(pink, template DNA;yellow, non-template DNA;gray, 10 element). NTPs and 6-mer RNA in the transcription complexes aredepicted bygreen circles. Positions of the RNAP active site ( red sphere) as well as template DNA transcription start site (TSS, 1,red circle) are indicated. DNAbasesindicatedby blackdashedcirclesare disordered in the crystal structures.In a standard formatdescribingRNAP promoter sequence, the transcription startsite,whichbasepairswiththefirstiNTPandencodesthe5 -endoftheRNAchain,isdesignated1. DNA positions downstreamfrom the transcriptionstart siteincrease as positive numbers from1 andDNA positionsupstreamof thestart site increase as negative numbers counting from1. B, overall structureof thede novo transcriptioninitiation complex. T. thermophilus RNAP is depictedas a molecular surface model.Each subunit of RNAP is depicted with a unique color(white, ; cyan, ;pink, ; orange, ).DNA isdepicted as a sphere modelwiththe same colorscheme as described inA. Rightpanelshows a magnified viewofthe boxedregionin the left panel. Forclarity,the subunit wasmade transparent in theleft panel andremoved in therightpanel.Several keymotifs discussedin the text are highlighted (see alsosupplemental Movie S1).



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1237 ofE. coliRNAP (counterparts ofT. thermophilusresi-dues Lys-838, Lys-846, and His-999, respectively) are closeto the binding site of the first iNTP and that their substitutionsimpaired transcription (25, 26). To further analyze the func-tions of the amino acid residues interacting with the first iNTP,we made four E. coli RNAP mutants, including Lys-1065Aand Lys-1065A/Lys-1073A, for residues interacting with-phosphate, and His-1237A and His-1237A/Gln-688A,for residues interacting with -phosphate, and tested theirtranscription activities on the T7A1 promoter (Fig. 3). The

mutations resulted in major defects in transcription initiationin the presence of low concentrations of NTPs, resulting indecreased abortive and full-length RNA synthesis (Fig. 3B,lanes 16). The activities of the Lys-1065A and His-1237ARNAPs were partially recovered by adding an initiating dinu-cleotide primer (lanes 712) and were further rescued in thepresence of high concentrations of NTPs (lanes 1318 and1924), suggesting that the mutations primarily affect the ini-tiation step of transcription. Importantly, the mutations alsoaffected the pattern of abortive products synthesized during

FIGURE 2. Firstand secondiNTPbindingsites ofthedenovo transcription initiation complex.A, activesite structureof thedenovo transcription initiationcomplex. DNA, iNTPs, andaminoacid side chainsare shown as stick models,and thetrigger loop, DFDGD motif,and theregion3.2(3.2) areshownas wormmodels and labeled. The disordered region of the trigger loop is shown as adashed line.F

o F

celectron density maps (yellow mesh) showing ATP (first iNTP)

and CMPCPP (second iNTP) are superposed on the final model. B, the first iNTP (ATP) binds at the active site through multiple interactions, including basepairing with the1DNAbase(red dashed lines), base stacking with the1 purine base (gray dashed lines), water-mediated interactions (yellow dashed lines),andsalt bridgeswithside chains(cyandashed lines). Aminoacid side chainsinvolved in thesecond iNTP (CMPCPP)bindingare also indicated.The transcriptionstart site (TSS) is indicated. C, comparison of the holoenzyme-promoter DNA (Protein Data Bank code 4G7H, magenta), de novotranscription (blue), andtranscription elongation complexes (Protein Data Bank code 2O5J, grayandwhite). RNAP structures were superposed at their active site domains of the

subunit. The iand i1 sitesare indicated.Trigger loops ( residues 12221265) of these structures are shown as tube models. D, ATPand CMPCPP bound intheactivesite.TheaspartateresiduesoftheDFDGDmotifofthe subunitcoordinatingtheMg2 (yellow spheres)arelabeled.Thedistancebetweenthe3-OHof ATP and the -phosphate of CMPCPP is 5.4 as indicated.E, amino acid residues involved in the first iNTP binding are conserved in all cellular RNAPs.



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initiation (Fig. 3B; see below).The double mutant His-1237A/Gln-688A did not differ significantly from the His-1237ARNAP; however, the double mutant Lys-1065A/Lys-1073Awas essentially inactive at all conditions tested, indicating thatresidue Lys-1073A is essential for initiation. The results high-light the relevance of these residues during de novotranscrip-tion initiation and are consistent with their proposed roles inthe first iNTP binding.

Basedonthepreviousobservationsontheroleofregion3.2in iNTP binding (3, 27), we next inspected possible interactionsbetween this region and the iNTPs loaded in the active site. The

closest distance between the region 3.2 (Glu-324 side chain)and the -phosphate of theisite bound ATP is 10.5 ; at thisdistance, the region 3.2 is positioned near the upstream tem-plate DNA bases 3/4 (Fig. 2A). This rules out any directinteraction between this region and the first iNTP during denovoinitiation and suggests that its stimulatory effect on iNTPbinding is indirect.

From the structure, we noticed a base stacking interactionbetween the guanine template position1andthe i site boundATP(supplemental Movie S1); also the N-1 of the guanine baseandthe -phosphate of ATPare connected by a water molecule(Fig. 2B). The base stacking interaction would be minimized inthe case of template DNA containing a pyrimidine base at the

1 position (Fig. 4Aandsupplemental Movie S1). These addi-tional interactions may contribute to stable binding of the ATPduringde novotranscription. To test this hypothesis, we mea-sured the effects of a G-to-C substitution at the 1 templateposition on theK

mof iNTPs on the T7A1 promoter (the wild-

type template strand sequence 3-GTAG-5, the transcriptionstartsiteisunderlined,Fig.3A). We found that this substitutiongreatly increased apparent K

m values for both 1ATP and

2UTP. In fact, the effect for the 2UTP was even strongerthan for the ATP (78.8- and 8.9-fold changes compared withthe wild-type T7A1 promoter, respectively) (Fig. 4B). Thus, the

interaction between the

1 template base and the

1ATP maynotonlycontributetothe1ATPbinding,butalsostabilizethetemplate DNA strand and thus stimulate the 2UTP binding.To further test this hypothesis, we also measuredK

msforE. coli

RNAP with a region 3.2 deletion (513519), whichdecreases the iNTP binding due to destabilizing the templateDNA strand (20). Deletion ofregion 3.2 increased K

ms for

both 1ATP and 2UTP on the wild-type T7A1 promoter.However, the promoter substitution only slightly increasedK

ms

for the mutant RNAP (3- and 2.5-fold for1ATP and2UTP,respectively), suggesting that both theregion 3.2 deletion andthe 1 nucleotide substitution disturb the template strandconformation.

FIGURE3. Transcriptionactivities of wild-type andmutantRNAPs on the T7A1promoter.A,thesequencesofthewild-typeT7A1promoteranditsvariantsused in this study. Positions of the 35, 10 promoter elements, and the transcription start site (1) are indicated. The T7A1 promoter variant has a basesubstitution at the1 position (highlighted inyellow); theT7A1 consensus(T7A1cons) promoter contains the consensus10 element(TATAAT). B, transcrip-tion was performed in the presence of low (10 M allfourNTPswithaddition of-[32P]UTP) or high (100M ATP, GTP, CTP, and 10M UTP) concentrations ofNTPs with or without initiating primerCpA (25M), correspondingto positions1/1 of thepromoter. Thepositions of therun-off(RO)andabortive(ab)RNAproducts are indicated.



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Second iNTP Binding Site in the de Novo Transcription Initi-

ation ComplexThe second iNTP, CMPCPP in our crystalstructure, is positioned through base pairing with templateDNA and interacts with the subunit trigger loop and basicresidues (Arg-557, Arg-879, and Arg-1029) on the rim ofthe secondary channel (Fig. 2B). In the holoenzyme-promoterDNA complex, the trigger loop is in an open conformation andthe tip of the loop (residues 12381251) is disordered (8),

whereas in the elongation complex with an NTP substrate atthei1 site, the trigger loop is in the closed conformation andforms two trigger helixes without disordered regions (Fig. 2C)(1). In the de novo transcription initiation complex, eventhough the trigger loop is in a more closed conformation com-pared with the holoenzyme-promoter DNA complex, the mid-dle portion of the trigger loop (residues 12461251) is still dis-ordered. Met-1238 and Gln-1235 residues from theN-terminal-helix of the trigger loop reach out to interact withthe base and 3-OH of CMPCPP, respectively. However,because the trigger helix is not fully folded, there is no directinteraction of the triphosphate group of the second iNTP withArg-1239 and His-1242, causing it to attain a preinsertion

conformation (Fig. 2D). In particular, the -phosphate ofCMPCPP is 5.4 away from the 3-OH of ATP forming anonreactive state. The catalytic metal MgA is bound stably atthe active site through the aspartate triad (residues at 739, 741,and 743) of the DFDGD motif of the subunit. However, MgB

is too far away for being coordinated by the aspartate triad (Fig.2D) and is primarilycoordinated by the -and -phosphates of

CMPCPP. This nucleotide position is similar to the preinser-tion state observed in crystal structures of the T. thermophiluselongation complex with the inhibitor streptolydigin (1), theeukaryotic Pol II elongation complex containing GMPCPP(guanine-5[(,)methyleno]triphosphate) (28), and the PolII initially transcribing complexes containing short RNA(27 nt in length) and AMPCPP (adenine-5[(,)methyleno]triphosphate) (29). Similar to these structures,using the non-hydrolyzable CMPCPP for preparing the bacte-rial de novo transcription initiation complex in this study mightaffect thecoordination of the iNTP and metals at the active site,thus positioning thei1 nucleotide in the precatalytic confor-mation, which likely corresponds to a natural intermediate in

formation of the catalytically competent transcription inititia-tion complex. During de novo transcription initiation in thepresence of natural iNTP substrates, the trigger loop wouldthen adopt the completely closed conformation, forming thetrigger helixes to push the iNTP at the i1 site closer to the3-OH at the i site and resulting in the formation of the firstphosphodiester bond.

Structure of the Initially Transcribing Complex of RNAP

Holoenzyme Containing 6-Mer RNACrystal structures oftranscription complex containing short RNAs of 2 to 7 nt inlength have been determined for the Pol II system. However,these complexes were prepared by incubating Pol II or PolII/TFIIB with DNA template and synthetic RNA oligonucleo-tides (2931). Therefore, these complexes lack the triphos-phategroupatthe5-end of RNA that may play important rolesin the early stages of transcription such as DNA/RNA hybridstabilization, separation of RNA from template DNA afterreaching the full hybrid lengthand removal of the transcriptioninitiation factor from RNAP. To obtain the structure of an ear-ly-stage transcription complex containing the factor and thenatural form of RNA containing the 5-triphosphate group, weprepared the initially transcribing complex by in crystalloapproach. For this purpose, we soaked the holoenzyme-pro-moter DNA complex crystals with ATP, CTP, and UTP, result-ing in the synthesis of a 6-mer RNA (the template sequence is

3-GTGAGTGC-5, the transcription start site is underlined)(Fig. 1A). The structure was solved at 3 resolution, and itshowed a continuous electron density for the 6-mer RNAsynthesized along the template DNA (Fig. 5, A and B, andsupplemental Movie S1), indicating that T. thermophilusRNAP was active in the crystalline state and capable of tran-scribing RNA to the expected length.

The RNA transcript remains in the pretranslocated state,with its 3-end bound in thei1 site; however, the pyrophos-phate by-product is not visible, and the middle region of thetrigger loop is disordered (residues 12391253) (Fig. 5B). Fur-ther movement of the DNA/RNA hybrid to a post-translocatedstate may be constrained due to steric hindrance between the

FIGURE4. RoleofthebasestackinginteractionbetweenthefirstiNTPandtemplate DNA in transcription initiation.A, schematic representation ofthe base stacking interaction between the1templateDNApurinebaseandthe first iNTP purine base. (see alsosupplemental Movie S1).B, apparentK

m

values for the initiating substrates on the wild-type T7A1 promoter (1Cnontemplate sequence) and its variant (1G non-template sequence) forRNAPs containing wild-type or 513519 70 factors. The numbers inblueandboldfacetype show changes inK

mvalues for the T7A1 promoter variant

relative to the wild-type sequence. C, non-template DNA sequence aroundthetranscription start sites of human Pol I, which is adaptedfrom Ref. 35.Thetranscription start site is 1.D, sequence conservation of the non-templateDNA around transcription start site of human Pol II. The sequence logo was

prepared using experimentally determined non-redundant collection ofhuman Pol II promoters, for which the transcription start site (1) has beendetermined experimentally. The majority of human Pol II promoters contain1 pyrimidine and1 purine bases in the non-template DNA.



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5-endofRNAandthe region3.2(seebelow),andtheejectionofregion3.2mayberestrictedduetocrystalpacking.Abiggerlobeof electron density appears nextto the RNA 3-end(Fig.5Aandsupplemental Movie S1). This density is close to the rim ofthe secondary channel and overlaps with the temporary NTPbinding Esite defined earlier in Pol II (32), which is distinctfrom the preinsertion site (1).

The downstream duplex DNA in the initially transcribingcomplex is shifted 6 base pairsupstream due to RNA transcrip-tion, whereas the contact between the upstream10 DNA ele-

ment and the region 2 is maintained. Thus, initial RNA tran-scription renders nontemplate DNA bases from 3 to 6disordered due to scrunching of the nontemplate strand (Fig.1Aandsupplemental Movie S1).

Compared with the de novo transcription initiation complex,the initially transcribing complex with 6-mer RNA also revealschanges in the region 3.2 and the template DNA near the region 3.2 (Fig. 5C). In thede novocomplex, the template DNAbases at the 4/3 positions are flipped out to make contactswith the acidic residues of the region 3.2, whereas in the

FIGURE5. Theinitially transcribingcomplexof RNAPholoenzymecontaining 6-mer RNA.A, FoF

celectrondensitymap(yellow mesh)showing6-merRNA

and a NTP bound at the Esite of the initially transcribing complex. The final model of 6-mer RNA and amino acid side chains involved in NTP binding at theEsite are shown as stick models and labeled. The DNA is depicted as CPK model.B, active site structure. DNA, RNA, and amino acid side chains contacting the6-mer RNAare shown as stick models.Disordered regions of thetrigger loop (residues 12391253) andthe region3.2(3.2, residues 321327)are indicatedby dashed lines.Thetranscriptionstartsite(TSS)isindicated.C,comparisonofthede novotranscription(blue,templateDNA;orange,region3.2)andthe6-merRNA initially transcribing complexes (pink, template DNA; green, 6-mer RNA). RNAP structures were superposedon theactivesite domains of the subunit. D,model of anextended RNAtranscriptplacedin theRNA exitchannelexplainsrelease. Structuresof the initially transcribing(pink,templateDNA;green,6-merRNA;orange,factor) and the transcription elongation complexes (Protein Data Bank code 2O5J, gray, template DNA;red, RNA;cyan, incoming NTP) weresuperposed on the active site domains of the subunit. The iand i1 sites are indicated.E, effects of the E. coliRNAP mutations on abortive synthesis.Transcription was performed on the T7A1cons promoter at high NTP concentrations in the presence of the CpA primer. Positions of the run-off ( RO) and

abortive products are indicated. The profilesabovethe gel show distribution of the RNA products for the wild-type, K1065A, and H1237A RNAPs ( black,blue,andred, respectively).



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initially transcribing complex, DNA bases at correspondingpositions upstream of the active site base pair with the nascent6-mer RNA. The 5-end of the RNA transcript reachesregion3.2 causing the tip of this region (residues 321327) to becomedisordered (Fig. 5B). This is likely due to the charge repulsionbetween the acidic cluster of the region 3.2 and the5-triphosphate of RNA. When RNAP continues transcription,

the region 3.2 has to be pushed further to accommodate lon-ger RNA transcript extending toward the region 4.1, ulti-mately resulting inejection from the RNA exit channel (Fig.5D).

Overall, the DNA/RNA hybrid in this initially transcribingcomplex is a typical A-form duplex with no intrinsic tilting thatwas observed earlier in the Pol II initially transcribing com-plexes (Fig. 5D) (29, 31). As the DNA/RNA hybrid extends inlength, the initial contacts between RNAP and the triphosphategroup of the first iNTP are lost. The positively charged andpolar residues involved in interactions with the triphosphategroup of the first iNTP in the de novotranscription initiationcomplex (Gln-567, Lys-838, Lys-846, and His-999) are

now involved in interactions with internal phosphate andribose groups at positions3/4 of the nascent RNA, suggest-ing their role in stabilization of the short RNA-DNA hybrid(Fig. 5B). To test the role of these residues in the extension ofshort RNAs, we analyzed abortive transcription by the wild-type and mutant E. coli RNAP variants on a consensusT7A1cons promoter (Fig. 3A), which is characterized by a veryhigh efficiency of abortive synthesis due to strong RNAP-pro-moter interactions (20). Transcription was performed at highNTP concentrations and in the presence of the dinucleotideprimer, to ensure efficient de novo initiation by the mutantRNAPs. Surprisingly, substitution Lys-1065A decreased abor-tive RNA synthesis (Fig. 5E), suggesting that this residue maydirectly or indirectly disturb short RNA extension in the wild-type RNAP, whileplaying a role in the firstbond formation (seeabove). In contrast, substitutions His-1237A and His-1237A/Gln-688A greatly stimulated the synthesis of10-ntabortive RNAs, but did not affect longer abortive products.Similarly,thesemutations increased theamounts of short abor-tive RNAs synthesized on the wild-type T7A1 promoter (Fig.3B, lanes 11and 12 and lanes 23 and 24). Therefore, residueHis-1237 plays an important role in stabilization of short nas-cent RNAs in the RNAP active site, until the full DNA/RNAhybrid is formed and the RNA reaches the RNA exit channel.

DISCUSSIONIn this study, we report the structures of bacterial transcrip-

tion initiation complexes with iNTPs or short RNA productbound in the active site of RNAP. In comparison with existingstructures, they reveal essential interactions of the iNTPs withtemplate DNA and for the first time highlight initial steps ofRNA extension by bacterial RNAP. Furthermore, we providebiochemical support to our structural findings and demon-strate the importance of the observed contacts of iNTPs andRNA with RNAP and the DNA template for transcriptioninitiation.

The binding of the first iNTP is a special feature of all cellularRNAPs that are capable of primer-independent de novotran-

scription initiation. A network of salt-bridge and hydrogenbonds between the triphosphate of the first iNTP and RNAPside chains is critical for its binding. At the same time, the posi-tions of the ribose and the base of the first iNTP are identical totheisite in the elongation complex. Furthermore, the positionof the second iNTP in the promoter complex corresponds tothepositionoftheincomingNTPboundinpreinsertionstatein

the elongation complex, indicating that the mechanisms of thephosphodiester bond formation are identical in the transcrip-tion initiation and elongation. Similar positions of the iNTPswere observed in theT. thermophilusRNAPde novotranscrip-tion initiation complex structure, which has been reported byEbright and co-workers (9) as a part of the analysis of theGE23077 antibiotic targeting bacterial RNAP while our manu-script was in preparation. We now provide strong biochemicalevidence in support of the importance of the observed iNTPcontacts with RNAP and DNA for de novo transcriptioninitiation.

The structures we obtained by using template DNA contain-ing a purine nucleotide at the 1 position revealed a stackinginteraction between the 1 purine base in template DNA andthe first purine iNTP. These stacking interactions were notobserved in previously reported structures because they used asuboptimal template sequence with a template pyrimidine at1 position. We showed that the base stacking interactionplays major roles in the binding of not only the first iNTP butalso the second iNTP, likely as a result of template stabilization(Fig. 4B). This purine-purinebase stacking during de novo tran-scription initiation explains the preference of the purine nucle-otide at the transcription start site and pyrimidine nucleotide atthe preceding position in the non-template strand in bacterialpromoters (Fig. 4A) (24, 33, 34). For example, in theMycobac-

terium tuberculosis genome, the occurrences of pyrimidinesand purines at the 1 and 1 non-template DNA bases are70 and 80%, respectively (33). It remains to be establishedwhether the presence of suboptimal 1 nucleotides in theminority of promoters has any specific role in transcriptionregulation in bacteria. The preferable pyrimidine (1) andpurine (1) combination is also found in the majority ofeukaryotic Pol I (35) and Pol II promoters (Fig. 4, CandD) (36).A similar base pair stacking interaction was noticed in thestructure of the N4 phage RNAP de novotranscription initia-tion complex (7), indicating that this mechanism may beuniversal.

Although the role of the factor in promoter recognition iswell established, thestructuralbasis for its rolein thebinding ofiNTPs remained unknown. Our structure reveals the absenceof any direct contacts between the region 3.2 and the firstiNTP. Rather, this region seems to guide the path of templateDNA, thereby positioning the DNA bases for ideal binding withtheiNTPs. Therefore,deletion of theregion3.2 likelydisturbstemplate DNA binding, especially around the transcriptionstart site, allowing it to adopt a floppy conformation that ham-pers stable binding of the iNTPs (3, 20, 37). The eukaryotic PolIIcounterpartoftheregion3.2istheB-readerofTFIIB,whichanalogously inserts its -helix deep into the DNA binding cleftclose to the active site of Pol II to ensure proper template DNA



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positioning as well as participate in the transcription start siteselection (30, 38, 39).

When RNAP transcribes a 6-mer RNA, the RNA 5-endreaches the tip of the region 3.2, causing it to become disor-dered, most likely due to repulsion between the RNA5-triphosphate and the acidic cluster at theregion 3.2 tip.The 5-triphosphate group is also partially disordered, indicat-

ing that there are no stable interactions between the region3.2 and RNA. Our initially transcribing complex structure indi-cates that synthesis of a short RNA transcript is the first step ofthe ejection process that is likely followed by ejection ofregion 4 from the RNA exit channel and allows for the initia-tion-to-elongation transition. Accordingly, deletion of the factor from its region 3.2 to C terminus eliminates abortiveRNAs (40), whereas deletion of the region 3.2 reduces only59-mer abortive RNAs (20). Similarly, the B-reader of factorTFIIB in eukaryotic Pol II should be extruded from the RNAchannel after RNA reaches 6 nucleotides in length, revealingstriking similarities to the bacterial system (23).

The initially transcribing complexes of RNAP holoenzyme

containing short RNAs, usually 212 nucleotides in length, areunstable in solution resulting in abortive initiation (10, 11). Inthis study, we demonstrated that the T. thermophilusRNAP isactive in crystallized state and is capable of synthesizing up to6-mer RNA. In the crystallized state, RNAP motions arerestricted by the crystal packing, likely stabilizing the initiallytranscribing complex and allowing for preparation of highlyhomogenous complexes. Thein crystallotranscription systemcan be used to study RNA transcription by Raman crystallogra-phy and time-resolved trigger-freeze crystallography. Analo-gous to the studies performed on single-subunit RNAPs (41,42), these new experimental approaches may allow to traceevents in not only initial transcription but also during the NTPaddition cycle in cellular RNAPs.

AcknowledgmentsWe thank Drs. Irina Artsimovitch and Shun-ichi

Sekine for providing information of culturing T. thermophilus cells

and RNAP purification and for plasmids. We thank the staff at Penn

State Fermentation Facility for supporting T. thermophilus cell cul-

ture and the MacCHESS for supporting crystallographic data collec-

tion. We thank Dr. Lilian Hsu for critical reading of the manuscript.

Figs. were prepared using PyMOL.

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