X-ray crystal structures elucidate the nucleotidyltransfer reaction of transcript initiationusing two nucleotidesMichael L. Gleghorna,1, Elena K. Davydovab,2, Ritwika Basua, Lucia B. Rothman-Denesb,3, and Katsuhiko S. Murakamia,3

aDepartment of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802; and bDepartment of Molecular Geneticsand Cell Biology, University of Chicago, Chicago, IL 60637

Edited* by E. Peter Geiduschek, University of California at San Diego, La Jolla, CA, and approved December 30, 2010 (received for review November 6, 2010)

We have determined the X-ray crystal structures of the pre- andpostcatalytic forms of the initiation complex of bacteriophage N4RNA polymerase that provide the complete set of atomic imagesdepicting the process of transcript initiation by a single-subunitRNA polymerase. As observed during T7 RNA polymerase transcriptelongation, substrate loading for the initiation process also drives aconformational change of the O helix, but only the correct basepairing between the þ2 substrate and DNA base is able to com-plete the O-helix conformational transition. Substrate binding alsofacilitates catalytic metal binding that leads to alignment of the re-active groups of substrates for the nucleotidyl transfer reaction.Although all nucleic acid polymerases use two divalent metals forcatalysis, they differ in the requirements and the timing of bindingof each metal. In the case of bacteriophage RNA polymerase,we propose that catalytic metal binding is the last step before thenucleotidyl transfer reaction.

DNA-dependent RNA polymerases (RNAPs) transcribe DNAgenetic information into RNA and play a central role in gene

expression. RNAP catalyzes a nucleotidyl transfer reaction, whichis initiated by the nucleophilic attack of an O3′ oxyanion at theRNA 3′ terminus to the α-phosphate (αP) of the incomingnucleotide, resulting in phosphodiester bond formation and re-lease of pyrophosphate (PPi). Both single-subunit T7 phage-likeRNAPs and the multisubunit cellular RNAPs possess two nucleo-tide-binding sites for loading the RNA 3′ end (P site) and theincoming NTP (N site) (1, 2). A two metal-ion catalytic mechan-ism has been proposed, as the enzyme possesses two divalentcatalytic and nucleotide-binding metal cations chelated by two orthree conserved Asp residues (3). The catalytic metal is a Lewisacid, coordinating the RNA 3′-OH lowering its pKa and facilitat-ing the formation of the attacking oxyanion. The nucleotide-bind-ing metal is coordinated by the triphosphate of the incomingnucleotide and stabilizes a pentacovalent phosphate intermediateduring the reaction. Both metal ions are proposed to have octa-hedral coordination at physiological Mg2þ concentrations (4).

During transcript elongation, RNAP carries out the loading ofa single nucleotide substrate at the N site followed by a nucleo-tidyl transfer reaction with the RNA 3′ end at the P site; this cycleis repeated as elongation proceeds. X-ray crystal structures of thesingle-subunit T7 phage RNAP (2, 5) have depicted the processof transcript elongation in detail and reveal a conformationalchange of the Fingers subdomain during substrate loading to theactive site as also observed in the A family of DNA polymerases(DNAPs) (6, 7).

Initiation is the only step in the entire transcription processwhere two nucleotide substrates are loaded at the active sitefollowed by a nucleotidyl transfer reaction. Compared with elon-gation, the process of initiation has not been well characterized byX-ray crystallography. An X-ray crystal structure of T7 RNAPinitiation complex was reported (8), but it was captured by usinga substrate analog 3′-deoxyGTP (Fig. S1B). This analog lacks theessential O3′ required for nucleotidyl transfer and catalytic metalcoordination resulting in the absence of the catalytic metal ion

in the structure and misalignment of the reactive groups of sub-strate. In the present study, we have used X-ray crystallographyand a natural substrate plus a proper substrate analog to capturea set of atomic resolution snapshots, from nucleotide bindingto nucleotidyl transfer reaction, (Fig. 1A and Table S1) to eluci-date a complete picture of the process of transcript initiationby the central domain of N4 phage virion-encapsulated RNAP(mini-vRNAP).

ResultsDesign of the X-Ray Crystallographic Experiment to Monitor the For-mations of Transcript Initiation Complexes. Previously, we reportedthe X-ray crystal structure of the binary complex (BC) of promo-ter DNA and N4 mini-vRNAP (9), which is a member of theT7-like single-subunit RNAP family (10) that recognizes a speci-fic DNA hairpin sequence with a 5-bp stem, 3-nt loop as itspromoter (Fig. 1B) (11–13). In the BC structure, from −1 to þ2template DNA bases point toward the nucleotide entry pore,whereas the þ3 template DNA base is flipped in the oppositedirection providing an opportunity to analyze the structural tran-sitions of DNA template bases at the þ1 and þ2 positions andof the enzyme upon nucleotide loading.

The structures reported in this study represent the precatalytic[substrate complex I (SCI); substrate complex II (SCII); mis-match complex (MC)] and postcatalytic [product complex (PC)]stages of transcript initiation (Fig. 1A). Each complex comprisesthe 120 kDa N4 mini-vRNAP and a 36-nt DNA, which includesthe P2 promoter 7 bp stem, stable and well-ordered 3-nt loophairpin followed by five bases of single-stranded DNA includingthe start site (þ1) (Fig. 1B). Promoter and template DNA regionsto þ3 ∼ 4 were well resolved in the crystal structures, but werecompletely disordered downstream. The P2_7a DNA sequenceof the transcription start site is CC at positions þ1 and þ2, toform Watson–Crick base pairs with two molecules of GTP uponnucleotide loading, followed by a nucleotidyl transfer reactionto produce a 2-mer RNA—5′-pppGpG-3′—and a leaving PPi.There are two molecules in the asymmetric unit and, in the casesof the SCI, SCII, and MC, both molecules are quasi-identical. In

Author contributions: L.B.R.-D. and K.S.M. designed research; M.L.G., E.K.D., R.B., andK.S.M. performed research; M.L.G., E.K.D., R.B., L.B.R.-D., and K.S.M. analyzed data;and M.L.G., L.B.R.-D., and K.S.M. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

Data deposition: The atomic coordinates and structure factors have been deposited in theProtein Data Bank, www.pdb.org (PDB ID codes 3Q22 for substrate complex I, 3Q23 forsubstrate complex II, 3Q0A for the mismatch complex, and 3Q24 for the product complex).1Present address: Department of Biochemistry and Biophysics, School of Medicine andDentistry, University of Rochester, Rochester, NY 14642.

2Present address: Department of Chemistry, University of Chicago, Chicago, IL 60637.3To whom correspondence may be addressed. E-mail: kum14@psu.edu or lbrd@uchicago.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1016691108/-/DCSupplemental.

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the case of PC, we observed a clear electron density map corre-sponding to the 2-mer RNA from only one complex (moleculeA); the other complex (molecule B) contained weak and discon-tinued map for the 2-mer RNA, suggesting that the 2-mer RNAwas partially dissociated from molecule B. We used molecule Afor the representative structure of transcript initiation.

All complexes were prepared by soaking GTP or its nonhydro-lysable analog, guanosine-5′-[(α,β)-methyleno] triphosphate(GMPCPP) (Fig. S1 A and C), and divalent cations, Mg2þ orMn2þ, to the preformed BC crystals. The overall structure ofthe BC (9) and the initiation complexes determined in this studyresemble a canonical “cupped right hand”; the enzyme active siteis located at the bottom of this cup (Fig. 1C) and does not interactwith any neighboring molecules in the crystals. Indeed, we ob-served N4 mini-vRNAP-catalyzed RNA synthesis in crystallo thatproduced the 2-mer RNA product (described below), indicatingthat the enzyme was active and able to perform any requiredconformational changes in crystallo.

Each initiation complex structure was determined by rigidbody and restrained refinements by using the N4 mini-vRNAPBC (9) as an initial model. After refinements with the BC models

against the structure factors from the initiation complex crystals,we observed clear unbiased Fo − Fc electron densities aroundthe active site, which corresponded to nucleotides and metalsin the precatalytic complexes and a product 2-mer RNA plusPPi in the postcatalytic complex (Fig. 1 D–F). Compared tothe binary complex, the backbone structures of the initiationcomplexes are almost identical (0.40 ∼ 0.65 Å rmsd) except fordistinct deviations in the part of Fingers (residues 657–770,1.5 ∼ 3.5 Å rmsd) including the O helix (residues 666–678) andDNA bases from −1 to þ2 (Figs. 2 and 3, and Movie S1).

Structure of Substrate Complex I: Presence of TwoMetals at the ActiveSite Is Essential for Catalysis. Precatalytic SCI (Figs. 1 A and Dand 2B) was prepared by soaking 5 mM GTP and 10 mM MgCl2into the BC crystals. SCI contained two molecules of GTP thatbase pair with DNA bases þ1 and þ2, and one Mg2þ ion as thenucleotide-binding metal. Mg2þ octahedrally coordinated withligands that include three atoms of the nonbridging triphosphateoxygens of GTPðþ2Þ, two carboxylates of the conserved Aspresidues (D559 and D951), and the main-chain carboxyl groupof G560 in the metal-binding motifs A and C that are commonto the T7-like single-subunit RNAP family.

The binding of the two GTPmolecules and the Mg2þ to the BCtriggers several conformational changes of DNA, the O helix ofthe Fingers and side-chain residues of motifs A and C in the Palm(Fig. 3A and Movie S1). Y678 at the O helix C terminus moves4.3 Å to open the GTPðþ2Þ binding pocket and hydrogen bondswith the 2′-OH of GTPðþ2Þ. This movement is linked to a con-formational change of the O helix, which swings approximately

A

B

D E F

C

Fig. 1. The structure of the initiation complex. (A) A schematic representa-tion of the sequential processes during initiation of transcription. The BCcomprising RNAP and promoter DNA is depicted as “E,” and catalytic andnucleotide-binding metal ions are shown as MeA and MeB, respectively.(B) Sequences and secondary structures of the two DNA constructs usedfor crystallization. Regions highlighted by the gray boxes were disorderedin the crystal structures. Nucleotide-binding sites (þ1 and þ2) for transcriptinitiation are colored in red. (C) Overall structure of the SCII. N4mini-vRNAP isdepicted as a molecular surface model. The N-terminal domain, subdomains,andmotifs are labeled. The β-intercalating hairpin, Plug, Thumb, and N-term-inal two-thirds of Fingers have been removed from this view for clarity, andonly their outlines are shown. The promoter DNA and O helix of the Fingersare depicted by a pink tube and blue ribbon, respectively. (D–F) Electron den-sity maps showing nucleotides, 2-mer RNA, pyrophosphate, and metal ionsfound in the three initiation complexes. Fo − Fc electron density maps (blacknet) superimposed on the final models (sticks and spheres) of the SCI (D), SCII(E), and PC (F). These maps were calculated using the native amplitudes andthe phase derived from the BC. Template DNA is depicted and labeled. Themetal-chelating D559 and D951 are shown as stick models. Divalent metalsand waters are depicted by yellow and cyan spheres, respectively.

Fig. 2. Structures of active site, DNA, and nucleotides during transcript in-itiation. The main chains (ribbon models) of motifs A and C (red) and of theO helix (blue), and the main and side chains (stick models) involved in nucleo-tide andmetal binding in the BC (A), SCI (B), SCII (C), and PC (D). NTP binding Pand N sites are indicated as green and magenta circles in A. DNA template(from −1 to þ2, pink) and nucleotides at þ1 (green) and þ2 (magenta) posi-tions are shown as stick models. Divalent metals (Mg2þ or Mn2þ) are depictedby yellow spheres. Hydrogen bonds and salt bridges are depicted by blackdashed lines. Amino acid residues discussed in the text are labeled.

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8° away from the active site. DNA template bases from −1 to þ2change their positions (−1∶1.4 Å, þ1∶3.0 Å, þ2∶2.1 Å) to bindGTPs at the P and N sites. The metal-coordinating carboxylatesD559 (motif A) and D951 (motif C) rotate their side chains tochelate the nucleotide-binding metal. The triphosphate ofGTPðþ2Þ forms extensive interactions with Y612, R666, andK670 in the Fingers and their side chains move their positionsupon GTPðþ2Þ binding. In our structural analysis, we identifieda conformational change of RNAP that is important for elicitinga proper environment for phosphodiester bond formation(Figs. 2 and 3A, and Movie S1). Although the conformationalchange of the O helix has been well characterized during sub-strate loading and catalysis of transcript elongation, our observa-tion clearly proves that the single-subunit RNAP indeed changesits conformation during the initiation process.

To investigate whether correct vs. incorrect base pairingbetween nucleotide and DNA template is critical for the O-helixconformational change, we determined the structure of an MCprepared by using BC crystals with P2_7c DNA (Fig. 1B) whichwere soaked with GTP plus MgCl2. In this setup, only a Watson–Crick base pair forms at theþ1 position but theþ2 position has aG (substrate)-T (DNA template) mismatch. In the MC structure,there is a clear density map corresponding to þ1 GTP that formsa Watson-Crick base pair with the þ1C DNA base; however, theþ2 substrate binding site shows only a subtle density (Fig. S2)likely reflecting the formation of an unstable mismatch betweenGTP andþ2 base of DNA template. The mismatch atþ2 positionis still able to trigger the conformational change of the O helixand Y678, but their positions deviate from the ones observedin the SCI structure (Fig. 3F), indicating that only the correctbase pairing between þ2 substrate and DNA base is able to com-plete the O-helix conformational transition. A partially changed

O-helix conformation may function as an intermediate kineticcheckpoint for substrate discrimination and also relate to aunique species, between the open and closed O-helix conforma-tions, found in the mismatch complex of DNAP I by single-molecule FRET analysis (14).

In the SCI, there was no electron density corresponding tothe catalytic metal (Fig. 1D), possibly due to the presence ofcitric acid (0.11 M) in the crystallization solution, which formsa stable metal–ligand complex resulting in a decreased concen-tration of free Mg2þ. The stabilization constant (log10 K,K ¼ ½ML�∕½M�½L�, metal, M; ligand, L) between Mg2þ and citricacid is 2.8, which is larger than that between Mg2þ and asparticacid (2.43) (15). The significantly larger stability constant ofMg2þ and nucleoside triphosphate (4.0) allowed the coordinatednucleotide-binding metal in the SCI to be retained. Other exam-ples of Mg2þ chelation by citric acid in crystal structures, whichprevented Mg2þ binding to the catalytic metal site, have beenreported [e.g., DNAP λ (16) and CCA adding polymerase (17)].SCI possesses all of the components required for catalysis exceptfor the catalytic metal; its absence prevents the nucleotidyltransfer reaction even in the presence of reactive GTPs at theactive site. The result indicates that substrate loading drives theconformational change of the O helix, although it is not sufficientfor nucleotidyl transfer, and that the presence of both metal ionsat the active site is essential for catalysis (3).

The GTPðþ1Þ and GTPðþ2Þ Binding Sites. In the case of transcriptinitiation, a single nucleotide has to be positioned at the P siteprior to the first nucleotidyl transfer reaction, and a single basepair with the template DNA is most likely not sufficient forGTPðþ1Þ binding. The SCI structure revealed extensive interac-tions between the GTPðþ1Þ triphosphate and two basic residues—K437 and R440—in the Palm core (Fig. 2B). These interac-tions, which are unique to initiation, because only at this stageis a nucleoside triphosphate loaded at this position, may compen-sate for the weaker binding of GTPðþ1Þ. Accordingly, K437A-and R440A-substituted enzymes had lower affinities for the initi-ating nucleotide (Km ¼ 200 and 100 μM for K437A and R440A,respectively, vs. 50 μM for the wild-type enzyme) and significantlyreduced in vitro transcription activities compared with the wild-type enzyme in the presence of NTP at low concentration(4 μM); higher NTP concentration (500 μM) partially restoredthe activity of the R440A enzyme but not of the K437A enzyme(Fig. 4A). These results suggest that K437 and R440 play a rolein nucleotide binding for transcript initiation and K437 plays amore important role than R440. To ascertain the site of transcriptinitiation by the mutant enzymes, we cross-linked the hydro-xybenzaldehyde ester of GTP to the enzyme; addition of[α − 32P]ATP led to phosphodiester bond formation in a tem-plate-directed manner and enzyme autolabeling (10). Catalyticautolabeling of the mutant enzymes at high-NTP concentrationconfirmed that initiation occurred at position þ1 (Fig. 4B).

The BC structure revealed that residue R318 in the N-terminaldomain forms a cation-π interaction with DNA base −2 andsalt bridges with the phosphate backbone that induce a DNA kinkbetween bases −2 and −1. During substrate loading, the −1DNAbase changes its position to partially stack with GTPðþ1Þ in theinitiation complexes (Fig. 2 A and B). The stacking of purinebases between −1 DNA base and GTPðþ1Þ may facilitateGTPðþ1Þ loading at the active site. This combination, a purineat position −1 on the template strand and at position þ1 on thenontemplate strand, is also found in the majority of Escherichiacoli σ70-dependent promoters (18), which is consistent with thehypothesis that the −1 template base plays a similar role in initialNTP binding by the bacterial RNAPs.

GTPðþ2Þ is located at the N site (Fig. 2B) and has a uniquebase-specific hydrogen bond between the keto group of guano-sine and the N671 side chain, which is positioned in the middle

+1

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A R666

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Bγγ5.9 Å(57°) R666

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Fig. 3. Structural transitions of the active site, DNA, and nucleotides asso-ciated with transcript initiation. Superposition of the BC and SCI structures(A), SCI and SCII structures (B), and SCII and PC structures (C) showing theconformational changes induced by nucleotide and metal binding and thenucleotidyl transfer reaction. BC, SCI, SCII, and PC are colored in black, yellow,green, and orange, respectively. The O helix is depicted as a ribbon model.DNA template (from −1 to þ2, pink), nucleotides, and amino acid side chainsinvolved in nucleotide and metal bindings are shown as stick models andlabeled. Divalent metals (Mg2þ or Mn2þ) are depicted by spheres, and cata-lytic and nucleotide metals are indicated as “A” and “B,” respectively. Hydro-gen bonds and salt bridges are depicted by yellow (in SCI), green (in SCII),and orange (in PC) dashed lines. Close-up views of reactive groups—O30ðþ1Þ and αPðþ2Þ—of SCI and SCII structures (D) and SCII and PC struc-tures (E). In D, the distance between O30ðþ1Þ and αPðþ2Þ is reduced upon thecatalytic metal binding. In E, the [O30ðþ1Þ-catalytic metal-αPðþ2Þ] angle ischanged from 84° to 49° by phosphodiester bond formation. (F) Superposi-tion of the BC, SCI, and MC structures showing the partial conformationalchange of O helix found in the MC. BC, SCI, and MC are colored in black,yellow, and pink, respectively. This view is the same as in A.

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of O helix. This interaction stabilizes the binding of GTPðþ2Þbecause the N671A enzyme had decreased affinity for the secondnucleotide (Km ¼ 120 μM for the N671A enzyme vs. 50 μMfor the wild-type enzyme) and reduced activity in the presenceof low NTP concentration (4 μM), which was restored to approxi-mately 80% at high-NTP concentration (500 μM) with initiationat þ1 (Fig. 4A).

The interaction between an amino acid residue at the middleof the O helix and nucleotide þ2might be universal for transcriptinitiation in the T7-like single-subunit RNAP family. These en-zymes contain amino acids with longer side chain with a hydro-philic moiety (Arg, Lys, Asn, or Gln) at this position, whichare capable of making base-specific interactions (Fig. S3, Upper).T7 RNAP has a strong sequence preference for GTP at positionsþ1 and þ2 and has Arg at this position, which is capable of mak-ing base-specific contact with the 6-keto and/or the 7-iminogroups of GTP at position þ2. Mitochondrial RNAP, which hasa strong sequence preference for ATP or GTP at þ2, possessesGln at this position, which is able to be a hydrogen donor andacceptor at this position. The bifunctional character of Glnmay allow this side chain to establish a hydrogen bond withthe 7-amine group of ATP and 6-keto group of GTP. This extrainteraction between the þ2 NTP base and amino acid side chainof the O helix enhances formation of the first phosphodiesterbond. However, it may decrease the fidelity of nucleotide selec-tion during transcript elongation by increasing the affinity of theincorrect NTP at active site. Accordingly, the A-family DNAPs,which require a preexisting primer for catalysis, contain a rela-tively short side-chain residue at this position (Fig. S3, Lower).Furthermore, a substitution of T664 with Arg in Thermus aqua-ticus DNAP I reduced its specific activity about threefold andincreased the mutation frequency about 25-fold (19), suggestingthat DNAPs have most likely eliminated the interaction betweenthe amino acid residue at the middle of the O helix and dNTPat the N site in order to enhance DNA replication fidelity.

The ribose ring of GTPðþ1Þ is in the C3′-endo conformationand its O30ðþ1Þ is in line with αP and the leaving bridging oxygen

between αP and βP of GTPðþ2Þ (Fig. 3D). However, the distancebetween O30ðþ1Þ and αPðþ2Þ is 4.1 Å, which is distinctly longerthan distances (3.3 ∼ 3.7 Å) reported from other precatalyticforms of polymerase structures, including T7 RNAP in the elon-gation complex (2) and X-family DNAPs (16, 20). This configura-tion distance indicates that the geometry of the reactive groups—O30ðþ1Þ and αPðþ2Þ—in the SCI may not be competent forcatalysis and suggests that catalytic metal binding at the site willrealign these groups for phosphodiester bond formation (4, 21).

Structure of Substrate Complex II: Loading the Catalytic Metal to theActive Site Induces Conformation Changes of the Enzyme Active Siteand Nucleotide þ1. To load the catalytic metal but prevent phos-phodiester bond formation, we soaked 20 mM MnCl2 and 5 mMof GMPCPP into the preformed BC crystals. The stability con-stant of the Mn2þ-aspartate complex (log10 K ¼ 3.74) is higherthan its citric acid counterpart (log10 K ¼ 2.8) (15) allowingMn2þ binding at both sites. Mn2þ has octahedral coordinationwith almost identical metal-donor distances as those observedwith Mg2þ (22). In addition, both Mg2þ and Mn2þ can activatecatalysis in vitro by N4 vRNAP (Fig. S4) and other members ofthis type of polymerase including T7 RNAP (23) and E. coliDNAP I (24). The structure was determined at 1.8-Å resolutionwith clear unbiased Fo − Fc electron densities around the activesite, corresponding to two molecules of GMPCPP and twoMn2þ ions (Fig. 1E). We termed this precatalytic complex SCII(Figs. 1A and 2C).

Loading the catalytic metal into the active site aligned thereactive groups of substrates and the catalytically essential car-boxylates for the nucleotidyl transfer reaction: (i) The O30ðþ1Þmoved in the direction of αPðþ2Þ and the distance betweenthe two groups decreased from 4.1 to 3.1 Å (Fig. 3D); and (ii) theD559 side chain also moved 1.8 Å to chelate both catalytic andnucleotide-binding metals (Fig. 3B), with the metals separated by3.6 Å. The catalytic metal binding induced an unexpected confor-mational transition of the triphosphate moiety of nucleotide þ1.Compared to the SCI structure, the γ-phosphate (γP) group ofGMPCPPðþ1Þ in SCII moved 5.9 Å toward the catalytic metal,thus becoming one of six ligands that coordinate the catalyticmetal (Fig. S5A). This drastic motion disrupted the interactionbetween R440 and the triphosphate, and established a new inter-action between the γP groupðþ1Þ and E557. To allow this inter-action, a nonbridging oxygen associated with γPðþ1Þ is most likelyprotonated (pKa value for secondary phosphate ionization inunbound nucleotide triphosphate is approximately 7.6) (25). Therelevance of this interaction was supported by the behavior of theE557A-mutant enzyme, with decreased runoff transcription activ-ity at 2 mM Mg2þ and some recovery at 10 mM Mg2þ concentra-tion, without a change in the site of initiation (Fig. 4 C and D).

To assess the role of the γP group ðþ1Þ in transcript initiation,we determined the kinetic parameters for GTP and GDP incor-poration at the RNA 5′ end (Table 1). N4 mini-vRNAP had afourfold higher affinity (50 μM) and a threefold higher kcat(300 min−1) for GTP than for GDP (200 μM and 100 min−1) atphysiological (1 mM) Mg2þ concentration. As a control, we usedT7 RNAP, which does not interact with the 5′ phosphate of theinitiating nucleotide (8, 26). Accordingly, the kinetic parameterswere identical when T7 RNAP initiated with GTP or GDP(200 μM and 20 min−1). Two charged residues—K437 andE557—are involved in positioning of the triphosphate groupðþ1Þ in contact with the catalytic metal. The functional rolesof K437 and E557 are supported by an analysis of the kineticparameters of GTP and GDP incorporation by the K437A andE557A enzymes. Both mutant enzymes show similar affinities(200 μM) for GTP and GDP as the initiating nucleotide. Notably,the affinity of the K437A and E557A enzymes for the initiatingnucleotide is similar to that of T7 RNAP (Table 1), whose activesite is superimposable with that of N4 vRNAP (27); however, T7

Fig. 4. Role of K437, R440, E557, and N671 residues in initiation of transcrip-tion by the mini-vRNAP. (A) Effect of K437, R440, and N671 substitutions onmini-vRNAP runoff transcription at increasing NTP concentrations. (B) Effectof Alanine substitutions at K437, R440, and N671 on selection of the site oftranscript initiation. Catalytic autolabeling was performed on templateswith increasing numbers (n) of As between the promoter hairpin and CTAwith increasing concentrations of the hydroxybenzaldehyde derivative ofGTP (bGTP). Awild-type vRNAP promoter contains 4As and initiates transcrip-tion 11 nt from the center of the hairpin at C. (C) Effect of Mg2þ concentra-tion on runoff transcription by E557A-mutant mini-vRNAP. (D) Effect ofE557A substitution on selection of the transcript initiation site. Catalyticautolabeling was performed as described in B at 1-mM bGTP.

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RNAP lacks residues equivalent to N4 vRNAP K437 and E557.The 15-fold decrease in kcat upon replacement of E557 by Alahighlights the relevance of E557 in catalysis of the N4 vRNAP.

Structure of the Product Complex: Phosphodiester Bond FormationReleases Both Metals from Their Binding Sites.The final step of tran-script initiation is the nucleotidyl transfer reaction yielding a2-mer RNA and PPi (Fig. 1A). To understand the structural basisof this chemical reaction, we attempted to carry out the nucleo-tidyl transfer reaction in crystallo and determine its structure. Wefound a unique electron density map at the active site in BC crys-tals with P2_7a DNA soaked with 0.5 mM GTP and 10 mMMgCl2 (Fig. 1F). There was a GTP-like structure at position þ1and a nucleotide at þ2 with a single phosphate group. The dis-tance between O30ðþ1Þ and αPðþ2Þ was 1.6 Å, indicating that thisdensity map corresponds to the 2-mer RNA (5′-pppGpG-3′)product. The PPi product, coordinated by residues K666, R660,and Y612, was also observed.

The 5′ and 3′ ends of RNA in the PC were found at the P andN sites, respectively (Fig. 2D), indicating that the PC was in apretranslocation state (2). The template DNA did not changeits position and the enzyme maintained the O-helix closedconformation with the Y678 side chain in the same position asobserved in the SCI and SCII (Fig. 3C and Movie S1). In thePC, weak electron densities were present at its metal-binding sites(Fig. 1F); however, the coordination distances of these densitieswere longer (density at catalytic metal site, 3.1 Å; density atnucleotide-binding metal site, 3.5 Å) than the expected distancefor Mg2þ (2.1 ∼ 2.3 Å), and their coordination spheres lacked theoctahedral geometry (22). We therefore assigned these densitiesas isoelectronic water, indicating that both catalytic and nucleo-tide-binding Mg2þ ions had dissociated after the nucleotidyltransfer reaction. The release of metal ions did not shift the2-mer RNA to the posttranslocated position or release PPi,but triggered conformational changes of the catalytic carboxy-lates (D557 and D951); their positions were approximately thesame as found in the BC, indicating that these residues formthe catalytically relevant conformation only in the presence ofmetals (Fig. 3C). In addition, metal release moved the tripho-sphate of nucleotide þ1 to a position nearly identical to thatobserved in SCI. The product PPi remained associated with theO helix through interactions with Y612, R666, and K670, butthese residues changed their positions to those found in the BC.

DiscussionTranscript Initiation by Single-Subunit T7 Phage-Like RNAPs.We havedetermined the high-resolution X-ray crystal structures of threedistinct forms of transcript initiation complexes during the forma-tion of 2-mer RNA, which revealed the formation of two inter-mediates—SCI and SCII—prior to the nucleotidyl transferreaction. In SCI, we observed the conformational change of theO helix upon binding of nucleotides þ1 and þ2 and nucleotide-binding metal (compare BC and SCI, Fig. 3A); nonetheless, thereactive groups—O30ðþ1Þ and αPðþ2Þ—do not possess the cat-alytically competent configuration. Binding of the catalytic metalresults in alignment of the substrates’ reactive groups to allow the

reaction to proceed (compare SCI and SCII, Fig. 3 B and D). Inthe PC, the catalytic metal is released after phosphodiester bondformation, indicating that the catalytic metal coordinatingO30ðþ1Þ and nonbridging oxygen of αPðþ2Þ in the 2-mer RNAcannot maintain octahedral coordination geometry (compareSCII and PC, Fig. 3 C and E). In other words, binding of the cat-alytic metal is sensitive to positions of these ligands that canbe easily influenced by correct vs. incorrect base pairing betweenthe nucleotide and DNA template base. Therefore, a small dif-ference in binding energy from correct vs. incorrect Watson–Crick base pairing is able to be converted into a large differencein catalytic efficiency; reactive groups are in an inactive config-uration and the 3′ oxyanion cannot be produced in the absenceof the catalytic metal, whereas they are properly aligned to gen-erate the 3′ oxyanion for catalysis in the presence of the catalyticmetal. Based on these facts, we propose that binding of the cat-alytic metal at the active site is the last step in the formation ofthe catalytically competent transcription complex and that cata-lytic-metal-dependent substrate alignment is the most criticalcheckpoint for fidelity of nucleotide incorporation by single-subunit RNAPs, and possibly by the A-family of DNAPs.

The published structure of the T7 RNAP transcript initiationcomplex (8) poses several problems: (i) Distances between thenucleotide-binding metal and its ligands are significantly greaterin this structure (average 4.3 Å) than in the elongation complex(average 2.7 Å) (2); (ii) although Y639 has been shown to discri-minate NTP against dNTP at the N site for both transcript initia-tion and elongation (28), Y639 does not contact the 2′-OH ofGTPðþ2Þ in the initiation complex structure; (iii) although theinteraction between H784 and the 2-amino group of GTPðþ1Þwas shown to play a role in transcription start site selection(29), the H784 side chain contacts GTPðþ2Þ in the structure; and(iv) no motion of RNAP or of the template DNA strand was ob-served during substrate loading at the active site (8). Therefore,we suspect that the proposed mechanism of transcript initiationbased on this T7 RNAP structure requires reevaluation. Further-more, the X-ray crystal structure of the T7 RNAP initiationcomplex (8) identified a unique nucleotide-binding site thatthe authors termed the D site (de novo site), which is distinct fromthe P site used for transcript elongation. In order to determinewhether the N4 vRNAP SCI possesses a D site for GTPðþ1Þ bind-ing, we superposed the N4 SCI with the T7 RNAP initiation (8)and elongation complexes (2) by overlaying their Palm cores in-cluding the T/DxxGR motif and motifs A and C (Fig. S6 A and B).BothGTPs at positionsþ1 andþ2 in the N4 SCI overlaid well withthe P and N sites of the T7 elongation complex, but not with theGTP binding sites found in the T7 initiation complex, indicatingthat N4 vRNAP does not use a D site for GTPðþ1Þ binding.

Transcript Initiation by Cellular RNAPs. All organisms have multisu-bunit RNAPs that carry out primer-independent transcript initia-tion. Crystallographic studies of cellular RNAPs have revealedinsights into the mechanism of transcript elongation (1, 30). How-ever, due to their larger size and complexity of preparation, X-raycrystal structures capturing transcript initiation with cellularRNAP have been elusive. In order to obtain structural insights

Table 1. Summary of N4 mini-vRNAP and T7 RNAP kinetic parameters for GTP and GDP

Initiation nucleotide

GTP GDP Enzyme catalyticefficiency GTP/GDP

Km, μM kcat, min−1 kcat∕Km, min−1 μM−1 Km, μM kcat, min−1 kcat∕Km, min−1 μM−1

Wild-type* 50 300 6.00 200 100 0.50 12.00K437A* 200 70 0.35 300 70 0.23 1.52E557A* 200 20 0.10 200 20 0.10 1.00T7 RNAP 200 20 0.10 200 20 0.10 1.00

Conditions as described in SI Experimental Procedures.*N4 mini-vRNAP.

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into the process of transcript initiation of cellular RNAPs, wecompared the coordination geometry of the catalytic metal ionin the N4 mini-vRNAP SCII structure with the γP group ðþ1Þinvolved in catalytic-metal coordination (Fig. S5A) and the Ther-mus thermophilus RNAP elongation complex structure (1), as arepresentative cellular RNAP because it is the highest resolutionstructure determined to date (Fig. S5B). The cellular RNAP usesthree carboxylates in the absolutely conserved 739-DFDGD-743motif in the largest subunit (region D) (31). The first Asp residuein the DFDGDmotif is involved in both catalytic- and nucleotide-metal coordination, whereas the second and third Asp residuescoordinate only the catalytic metal. The second Asp residue incellular RNAP localizes to the same position as the one occupiedby the γP group ðþ1Þ of the N4 RNAP SCII; however, a directcomparison of the two structures does not take into account theabsence of the triphosphate moiety at the P site nucleotide in theT. thermophilus RNAP elongation complex structure. The sixfolddecrease in Vmax observed for 2-mer RNA synthesis when ATP issubstituted by ADP as the initiating nucleotide in E. coli RNAPtranscription from the λ Pr promoter (32) might reflect the role ofthe γP group ðþ1Þ in transcript initiation by cellular RNAPs.Whether the second Asp residue plays a role in interacting withthe γP group ðþ1Þ in bacterial RNAPs awaits the determinationof the structure of their initiation complexes.

In the case of single-subunit RNAP, binding of the catalyticmetal requires the presence of template DNA and substratesat the active site, which is in contrast to the multisubunit RNAPsfrom Bacteria (31), Archaea (33), and Eukaryote (34), which co-ordinate the catalytic metal at the active site even in the absenceof DNA template or substrates. The difference between single-subunit and cellular RNAPs may reflect the fact that two andthree carboxylates are involved in coordinating the catalytic metalat the active site in single- and multisubunit enzymes, respectively

(Fig. S5). This structural difference may explain the fact that thesingle-subunit enzyme carries out only RNA synthesis, whereasthe multisubunit enzyme is capable of both RNA synthesis andRNA cleavage reactions, which play an important role in tran-scriptional proofreading and releasing arrested enzyme (35).

Experimental ProceduresDetailed protocols of (i) N4mini-vRNAP andDNA purifications,(ii) crystallization of binary complexes, (iii) preparing transcriptinitiation complexes, (iv) X-ray data collections and structure de-terminations, (v) site-directed mutagenesis of N4 mini-vRNAP,(vi) runoff transcription and catalytic autolabeling, and (vii) tran-script initiation assay and kinetics of first phosphodiester bondformation are described in SI Experimental Procedures.

Note. Recently, using fluorescence-based assays and stopped flow kinetics,Bermek et al. proposed a reaction pathway for E. coli DNAP I where theO-helix conformational change and the catalytic metal binding occur at earlyand late stages of the reaction, respectively (36). These stages coincide withthose we have defined based on the in crystallo reaction.

ACKNOWLEDGMENTS. We thank the staff at X25 of the National SynchrotronLight Source, F1 of the Macromolecular Diffraction Facility at Cornell HighEnergy Synchrotron Source (MacCHESS), and H. Yennawar for supportingcrystallographic data collection. We thank P.C. Bevilacqua, C.E. Cameron,P.R. Carey, Y. Chen, and R. Yajima for discussion, and S.J. Benkovic andT. Ellenberger for comments. We thank W. Ross and R.L. Gourse for criticalreading of the manuscript. Figures were prepared using PyMOL (http://pymol.sourceforge.net/). This work was supported by National Institutesof Health (NIH) Grants AI12575 and GM071897. The Cornell High EnergySynchrotron Source is supported by the National Science Foundation (NSF)and NIH/National Institute of General Medical Sciences via NSF awardDMR-0225180, and the MacCHESS resource is supported by NIH/NationalCenter for Research Resources award RR-01646.

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