Cell, Vol. 104, 901–912, March 23, 2001, Copyright 2001 by Cell Press

Structural Mechanism for Rifampicin Inhibitionof Bacterial RNA Polymerase

Mutations conferring Rif resistance (RifR) map almostexclusively to the rpoB gene (encoding the RNAP bsubunit) in every organism tested, including E. coli

Elizabeth A. Campbell,* Nataliya Korzheva,†Arkady Mustaev,† Katsuhiko Murakami,*Satish Nair,* Alex Goldfarb,† and Seth A. Darst*‡

*The Rockefeller University (Ezekiel and Hutchins, 1968; Wehrli et al., 1968b; Heiland Zillig, 1970) and M. tuberculosis (Ramaswamy and1230 York Avenue

New York, New York 10021 Musser, 1998; Heep et al., 2000). Comprehensive ge-netic analyses have provided molecular details of amino†Public Health Research Institute

455 First Avenue acid alterations in b conferring RifR (Figure 1; Ovchinni-kov et al., 1983; Lisitsyn et al., 1984a, 1984b; Jin andNew York, New York 10016Gross, 1988; Severinov et al., 1993; Severinov et al.,1994).

High-resolution structural studies of the Rif-RNAPSummarycomplex should lead to insights into Rif binding, themechanism of inhibition, and also the mechanism byRifampicin (Rif) is one of the most potent and broad

spectrum antibiotics against bacterial pathogens and which mutations lead to RifR. This could shed light onthe transcription mechanism itself, as well as provideis a key component of anti-tuberculosis therapy, stem-

ming from its inhibition of the bacterial RNA polymer- the basis for the development of drugs that selectivelyinhibit bacterial RNAPs but are less prone to singlease (RNAP). We determined the crystal structure of

Thermus aquaticus core RNAP complexed with Rif. amino acid substitutions giving rise to resistance. Therecent determination of the crystal structure of coreThe inhibitor binds in a pocket of the RNAP b subunit

deep within the DNA/RNA channel, but more than 12 A RNAP from Thermus aquaticus (Taq; Zhang et al., 1999)has opened the door to further studies of RNAP struc-away from the active site. The structure, combined

with biochemical results, explains the effects of Rif on ture, function, and interactions with substrates, ligands,and inhibitors. Here we describe the 3.3 A crystal struc-RNAP function and indicates that the inhibitor acts by

directly blocking the path of the elongating RNA when ture of Taq core RNAP complexed with Rif. The structureexplains the effects of Rif on RNAP function. In combina-the transcript becomes 2 to 3 nt in length.tion with a model of the ternary transcription complex(Korzheva et al., 2000) and biochemical experiments,Introductionthe data indicate that the predominant effect of Rif isto directly block the path of the elongating RNA tran-Each year, there are 8–10 million new cases of tuberculo-

sis (TB), which is the leading cause of death in adults script at the 59 end when the transcript becomes either2 or 3 nt in length.by an infectious agent (Raviglioni et al., 1995; Shinnick,

1996). With TB near epidemic proportions in some partsof the world and the rapid increase in multidrug-resistant Resultsstrains of Mycobaterium tuberculosis, the World HealthOrganization declared TB to be a global public health Rifampicin Inhibition of Taq RNAPemergency (Raviglioni et al., 1995). From a biochemical perspective, the interaction of Rif

Rifampicin (Rif; Sensi et al., 1960; Sensi, 1983) is one of with RNAP has been extensively characterized using E.the most potent and broad spectrum antibiotics against coli RNAP, which served as a prototype for bacterialbacterial pathogens and is a key component of anti-TB pathogens (Honore et al., 1993; Nolte, 1997; Rama-therapy. The introduction of Rif in 1968 greatly short- swamy and Musser, 1998; Drancourt and Raoult, 1999;ened the duration of TB chemotherapy. Rif diffuses Heep et al., 1999; Morse et al., 1999; Padayachee andfreely into tissues, living cells, and bacteria, making it Klugman, 1999; Wichelhaus et al., 1999). We investi-extremely effective against intracellular pathogens like gated the inhibition of Taq RNAP by Rif to assess thisM. tuberculosis (Shinnick, 1996). However, bacteria de- system as a structural model for Rif-RNAP interactions.velop resistance to Rif with high frequency, which has Sequence comparisons in the four distinct regions ofled the medical community in the United States to com- rpoB that harbor RifR mutations indicate a very high levelmit to a voluntary restriction of its use for treatment of of conservation among prokaryotes. Between E. coli,TB or emergencies. Taq, and M. tuberculosis, the sequences are 91% identi-

The bactericidal activity of Rif stems from its high- cal over 60 residues (93% conserved), explaining theaffinity binding to, and inhibition of, the bacterial DNA- broad spectrum of Rif activity. Nevertheless, among thedependent RNA polymerase (RNAP; Hartmann et al., 23 positions with single amino-acid substitutions that1967). The essential catalytic core RNAP of bacteria confer RifR in either E. coli or M. tuberculosis, 5 of these(subunit composition a2bb9v) has a molecular mass of (Taq b 387, 395, 398, 453, and 566; Taq numbering willaround 400 kDa and is evolutionarily conserved among be used throughout unless otherwise specified) are sub-all cellular organisms (Archambault and Friesen, 1993). stituted in Taq (Figure 1). In contrast, there is a relatively

low level of conservation between prokaryotes and eu-karyotes within these regions (Figure 1), explaining the‡ To whom correspondence should be addressed (e-mail: darst@

rockefeller.edu). lack of Rif activity against eukaryotic RNAPs.

Cell902

Figure 1. The Rif-Resistant Regions of the RNAP b Subunit

The bar on top schematically represents the E. coli b subunit primary sequence with amino acid numbering shown directly above. Gray boxesindicate evolutionarily conserved regions among all prokaryotic, chloroplast, archaebacterial, and eukaryotic sequences (labeled A–I at thetop; Allison et al., 1985; Sweetser et al., 1987). Red markings indicate the four clusters where RifR mutations have been identified in E. coli(Ovchinnikov et al., 1983; Lisitsyn et al., 1984a, 1984b; Jin and Gross, 1988; Severinov et al., 1993; Severinov et al., 1994), denoted as theN-terminal cluster (N), and clusters I, II, and III (I, II, III). Directly below is a sequence alignment spanning these regions of the E. coli (E.c.), T.aquaticus (T.a.), and M. tuberculosis (M.t.) RNAP b subunits. Amino acids that are identical to E. coli are shaded dark gray, and those thatare homologous (ST, RK, DE, NQ, FYWIV) are shaded light gray. Mutations that confer RifR in E. coli and M. tuberculosis are indicated directlyabove (for E. coli) or below (for M. tuberculosis) as follows: D for deletions, V for insertions, and colored dots for amino acid substitutions(substitutions at each position are indicated in single amino acid code in columns above or below the positions). Color coding for the aminoacid substitutions (for reference to subsequent figures) is as follows: yellow, residues that interact directly with the bound Rif (see Figure 4);green, residues that are too far away from the Rif for direct interaction (see Figure 5); purple, three positions that are substituted with highfrequency (noted as a % immediately below the substitutions) in clinical isolates of RifR M. tuberculosis (Ramaswamy and Musser, 1998).Below the three prokaryotic sequences is a sequence alignment of three eukaryotic sequences with shading as above. The dots indicate agap in the alignment.

A plate assay (see Experimental Procedures) showed RNAPs responded the same way, with an increase inthe production of the trimeric product and a concurrentthat Taq cells were unable to grow on media supple-

mented with 50 mg/ml Rif (data not shown). For in vitro precipitous drop in the production of the long transcripts(Figure 2a).studies, Taq RNAP holoenzyme was reconstituted using

Taq core RNAP (purified from Taq cells; Zhang et al., Mustaev et al. (1994) used chimeric Rif-nucleotidecompounds to measure the distance between the initiat-1999) and recombinant Taq sA (overexpressed and puri-

fied from E. coli; Minakhin et al., 2001b). The enzyme ing nucleotide binding site (the i-site) and the Rif bindingsite. By varying the linker between the Rif and the nucle-initiated, elongated, and terminated transcripts effi-

ciently from a template containing the T7A1 promoter otide and testing for maximal transcription initiation ac-tivity, the optimal length was found that allowed bindingand the tR2 intrinsic terminator (Figure 2a; Nudler et al.,

1994) at 378C using the dinucleotide CpA as the initiating of each moiety in its respective site. This experimentwas used to compare the disposition of the Rif andprimer. The major RNA products, a trimeric abortive

transcript (CpApU), a 105 nt terminated transcript i-sites in E. coli and Taq RNAP. In both cases, optimalinitiation activity was observed when the linker com-(Term), and a 127 nt runoff transcript (Run off), were the

same as those produced by E. coli RNAP (Figure 2a, prised five (CH2) groups (Figure 2b). Thus, in spite of thefact that Taq RNAP requires a 100-fold higher concen-lanes 1 and 8). Since E. coli s70 is totally inactive when

combined with Taq core RNAP in this assay (Minakhin tration of Rif for inhibition, we conclude that Taq RNAPbinds Rif and is inhibited through the same biochemicalet al., 2001b), the possibility of trace contamination with

E. coli s70 does not affect the conclusions from this assay mechanism as E. coli RNAP, and the disposition of theRif site with respect to the universally conserved activefor Taq RNAP. Quantitatively, the two RNAPs responded

very differently to Rif; the Ki (estimated from the Rif site is identical. We conclude that Taq RNAP can serveas a model for Rif interactions with other RNAPs.concentration where the production of long transcripts

was inhibited by 50%) for E. coli RNAP was about 0.1mM while for Taq RNAP it was about 10 mM, a 100- Rif-RNAP Structure Determination and Refinement

Tetragonal crystals of Taq core RNAP (Zhang et al.,fold difference in sensitivity (the Rif sensitivity of thethermophilic RNAP decreased at higher assay tempera- 1999) were incubated overnight in stabilization buffer

with 0.1 mM Rif, followed by a 30 min. soak in cryo-tures; data not shown). Qualitatively, however, both

Structure of the Rifampicin-RNA Polymerase Complex903

Figure 2. Rif Inhibition of Taq RNAP

(a) Autoradiographs showing the radioactive RNA produced by Taq (lanes 1–7) and E. coli (lanes 8–13) RNAP holoenzymes transcribing atemplate containing the T7 A1 promoter and the tR2 terminator, analyzed on a 15% polyacrylamide gel and quantitated by phosphorimagery.In the absence of Rif (lanes 1 and 8), the major RNA products from each RNAP correspond to a trimeric abortive product (CpApU), a 105 ntterminated transcript (Term), and a 127 nt runoff transcript (Run off). Lanes 2–7 and 9–13 show the effects of increasing concentrations ofRif. The quantitated results are shown on the right, where the amounts of each product (normalized to 100% for the Run off and Termtranscripts in the absence of Rif, and for CpApU at the highest concentration of Rif) are plotted as a function of Rif concentration.(b) The distance between the bound Rif and the initiating substrate (i-site) of E. coli and Taq RNAP holoenzymes was measured using chimericRif-nucleotide compounds as previously described (Mustaev et al., 1994). Rif-nucleotide compounds (Rif-(CH2)n-Ap) with different linker lengths,n (indicated above each lane), were bound to RNAP, then extended in a specific transcription reaction with a-[32P]UTP by the RNAP catalyticactivity. The products were analyzed on a 23% polyacrylamide gel, visualized by autoradiography, and quantitated by phosphorimagery. Thequantitated results are shown directly below, where the product yield (as % activity normalized to 100% at the highest level) is plotted as afunction of the Rif-nucleotide linker length (n).

Cell904

solution (without Rif) before flash freezing. During this and O10 are also in position to interact with the back-bone amide and carboxyl of F394, respectively. Alsoprocedure, the crystals took on a deep orange color,

confirming the binding of Rif. The same results were positioned to make a potential hydrogen bond with thebackbone amide of F394 is O8.obtained with cocrystals grown in the presence of 0.1

mM Rif, suggesting that Rif binding does not cause D396 contributes to the binding interface in severalways. In addition to forming a potential hydrogen bondsignificant conformational changes in the RNAP.

The Taq core Rif-RNAP crystals were isomorphous with O10, it forms the top end of the binding pocket (inFigure 4) by making van der Waals contact with C18–with the native Taq core RNAP crystals (Zhang et al.,

1999). Strong electron density was observed in differ- C21, and C31. Moreover, the negative charge of D396may be important for neutralizing the positive chargesence Fourier maps for the Rif (Figure 3a), which occupies

a shallow pocket between b structural domains 3 and of two nearby side chains, R405 (not shown) and R409(Figure 4), each about 6 A away. The charge neutraliza-4 (Figure 3b) that is surrounded by the known RifR muta-

tions (Figure 1; Zhang et al., 1999). The electron differ- tion might be important for the binding of the relativelyapolar Rif. Most RifR mutants at 396 substitute a large,ence density also indicated shifts and/or ordering of

several b residues interacting directly with Rif, including bulky group that would likely interfere with Rif bindingand would not have the correct geometry for hydrogenQ390, L391, Q393, D396, H406, R409, and L413 (Figure

4). Only very small shifts in localized regions of the pro- bonding O10 (Y), or else apolar groups (V, G, or A) with nohydrogen bonding ability. One of these mutants, D396Vtein backbone were indicated (not shown).

The Rif X-ray crystal structure (Brufani et al., 1974) was (position 516 in E. coli), was among the original, strongRifR mutants mapped by Ovchinnikov et al. (1983), point-easily placed into the difference density. Subsequent

refinements resulted in small shifts of the ansa chain ing to the importance of this residue in forming the Rifbinding interface. Another mutant identified in E. coli,(Figure 3c) to better fit the density. Multiple rounds of

manual rebuilding against (2|Fo| 2 |Fc|) maps and refine- however (D396N), is isosteric with Asp and would likelymaintain the hydrogen bond with O10. Nevertheless,ment resulted in the current model (see Experimental

Procedures and Table 1). this substitution yields weak RifR (Lisitsyn et al., 1984a),which may be caused by the loss of negative charge atthis position.Overall Structure

Rif has a partial 1 charge, localized at N4 (Figure 3c).As expected from the fact that all mapped RifR mutantsA negatively charged residue, E445, is situated nearbyoccur in rpoB (Figure 1), Rif makes contacts only withand may contribute to the Rif binding site by neutralizingthe RNAP b subunit in a close complementary fit to itsthis charge. This is not likely to be a strong effect, asbinding pocket deep within the main DNA/RNA channel.many Rif derivatives with equal or stronger activity thanClearly, Rif does not bind directly at the RNAP activeRif do not have this partial charge. E445 is the onlysite (Figure 3b). The closest approach of Rif to the activeresidue close enough to Rif to be involved in potentiallysite, defined as the distance between the active sitedirect interactions (Figure 4) for which a RifR mutant hasMg21 and Rif C38 (Figure 3c), is 12.1 A.not been reported. However, this residue is universallyconserved as either Glu or Asp in a segment of b regionDetailed InteractionsD that is invariantly present in prokaryotes, chloroplast,A large number of Rif derivatives have been investigatedarchaebacteria, and eukaryotes (Allison et al., 1985;for antimicrobial activity. In general, modification of theSweetser et al., 1987), pointing to its importance for theansa bridge, or modifications that alter the conformationbasic function of RNAP.of the ansa bridge, reduce activity. Other structural fea-

Thus, of the 12 residues that are close enough to Riftures of the antibiotic that are particularly critical forto make direct interactions (including backbone interac-activity include the napthol ring with oxygen atoms (O1tions with F394; Figure 4), 11 mutate to a RifR phenotype.and O2) at C1 and C8, and unsubstituted hydroxyls (O10The 12th position, E445, is highly conserved so that itsand O9) at C21 and C23 (Figure 3c; Brufani et al., 1974;substitution would likely be lethal and consequently notLancini and Zanichelli, 1977; Arora, 1981, 1983, 1985;be detectable as RifR mutations.Sensi, 1983; Arora and Main, 1984). Most Rif modifica-

Twelve additional positions have been identified attions that retain activity involve substitutions at C3 ofwhich substitution gives rise to RifR (Figure 1). Thesethe napthol ring, which have only modulatory effects onresidues surround the Rif binding pocket but do notin vitro activity.make direct interactions with the antibiotic (Figure 5).These results are explained by the structural detailsIn every case, the RifR mutations involve replacementof the Rif-RNAP complex (Figures 4 and 5). A cluster ofby a different sized amino acid side chain (almost alwayshydrophobic residues (L391, L413, G414, I452) line onesubstituting a small residue with a more bulky one), orwall of the Rif binding pocket and make van der Waalselse involve adding or removing a Pro residue. Thesecontact with the napthol ring and the methyl group atsubstitutions would likely affect the folding or packingC7. One end of the binding pocket (the bottom in Figureof the protein in the local vicinity of the substituted4) is formed by Q390. The alkyl chain of Q390 makesresidue, causing distortions of the Rif binding pocket.van der Waals contact with Rif C28 and C29, while the

polar head group may interact with O5. Protein groupsare positioned to make hydrogen bonds with each of Mechanism of RNAP Inhibition by Rif

The effects of Rif on RNAP in each stage of the transcrip-the four critical hydroxyls of Rif: R409 with O1, Q393and S411 with O2, and D396 and H406 with O10. O9 tion cycle have been probed using detailed kinetic analy-

Structure of the Rifampicin-RNA Polymerase Complex905

Figure 3. Rif-RNAP Cocrystal Structure

(a) Stereo view of the Rif binding pocket of Taq core RNAP, generated using O (Jones et al., 1991). Carbon atoms of the RNAP b subunit arecyan or yellow (residues within 4 A of the Rif), while carbon atoms of the inhibitor are orange. Oxygen atoms are red, nitrogen atoms are blue,and sulfur atoms are green. Electron density, calculated using (|Fo

Rif 2 Fonat|) coefficients (Rif denotes the Rif-RNAP cocrystal, native denotes

the native core RNAP crystal), is shown (orange) for the Rif only (contoured at 3.5 s), and was computed using phases from the final refinedRNAP model with the Rif omitted.(b) Three-dimensional structure of Taq core RNAP in complex with Rif, generated using GRASP (Nicholls et al., 1991). The backbone of theRNAP structure is shown as tubes, along with the color coded transparent molecular surface (b, cyan; b’, pink; v, white; the a subunits arebehind the RNAP and are not visible). The Mg21 ion chelated at the active site is shown as a magenta sphere. The Rif is shown as CPK atoms(carbon, orange; oxygen, red; nitrogen, blue).(c) Structural formula of Rif. Features of the structure discussed in the text are color coded (ansa bridge, blue; napthol ring, green). The fouroxygen atoms critical for Rif activity (Brufani et al., 1974; Lancini and Zanichelli, 1977; Arora, 1981, 1983, 1985; Sensi, 1983; Arora and Main,1984) are shaded with red circles.

ses. Rif has essentially no effect on specific promoter binding on RNAP activity was a total blockage of synthe-sis of the second (when transcription was initiated withbinding and open complex formation (Hinkle et al., 1972;

McClure and Cech, 1978). A small increase (about 2-fold) a nucleoside triphosphate) or third (when transcriptionwas initiated with a nucleoside di- or monophosphate)in the apparent Km for initiating substrate binding in

the enzyme’s i-site (the 59 nucleotide) was observed, phosphodiester bond (McClure and Cech, 1978). Sincesynthesis of the first and second phosphodiester bondsbut the binding of the incoming nucleotide substrate in

the i11 site (the 39 nucleotide) and the formation of can occur in the presence of Rif, the antibiotic does notinterfere with substrate binding, catalytic activity, or thethe first phosphodiester bond were largely unaffected

(McClure and Cech, 1978). The dominant effect of Rif intrinsic translocation mechanism of the RNAP. After

Cell906

Figure 4. Detailed Interactions of Rif with RNAP

(a) Stereo view of the Taq RNAP Rif binding pocket complexed with Rif, generated using RIBBONS (Carson, 1991), showing residues thatinteract directly with the inhibitor. The view is from the top of the RNAP in Figure 3b, above the b subunit, looking down through b to the Rif,but with obscuring parts of b removed. The backbone of the b subunit is shown as a cyan ribbon. Side chains (and backbone atoms of F394)of residues within 4 A of Rif are shown. Carbon atoms are orange (Rif), magenta (three residues substituted in M. tuberculosis RifR clinicalisolates with high frequency, see Figure 1), or yellow; oxygen atoms are red; nitrogen atoms are blue. Potential hydrogen bonds betweenprotein atoms and Rif are shown as dashed lines.(b) Schematic drawing of RNAP b subunit interactions with Rif, modified from LIGPLOT (Wallace et al., 1995). Residues forming van der Waalsinteractions are indicated, those participating in hydrogen bonds are shown in a ball-and-stick representation, with hydrogen bonds depictedas dashed lines. Carbon atoms of the protein are black, while carbon atoms of Rif are orange. Oxygen atoms are red and nitrogen atoms areblue.

RNAP has synthesized a long transcript and entered the elongating RNA at the 59 end (McClure and Cech, 1978).Whether Rif directly blocked the path of the RNA or ifelongation phase, it becomes totally resistant to Rif.

These properties led to the proposal that Rif inhibits blockage was an indirect effect due to a conformationalchange in the RNAP induced by Rif binding could notRNAP through a simple steric block of the path of the

Structure of the Rifampicin-RNA Polymerase Complex907

Table 1. Crystallographic Data and Structural Model

Diffraction Data

Parameter Total Outer Shell

Resolution range (A) 30–3.3 3.42–3.3Rmerge

a (%) 7.7 34.4Completeness (%) 86.1 71.1I/sI 10.7 1.7No. of reflections 75,420 6173No. of unique obs. 214,453 11,549

Structural Model

No. of ResiduesProteinSubunitb Mr (kDa) Sequence Model Regions Modeled

b9 170.7 1525 1139 3–31, 69–155 (poly-Ala), 452–523, 536–1241,1250–1410, 1414–1497

b 124.4 1119 1114 2–1115aI 34.9 313 223 6–228aII 34.9 313 229 3–231v 11.6 99 98 1–98Total 376.5 3369 2803

RefinementRcryst (%) 28.1Rfree (%) 35.9

a Rmerge 5 S|Ij 2 ,I.|/SIj.b Also included in the model was one Mg21 and Zn21 ion. (Zhang et al., 1999), and one Rif molecule (Brufani et al., 1964).

be distinguished. Alternatively, others have proposed transcript length of 3 nt, however, the 59 phosphates ofthe 59 nucleotide (at 22) sterically clash with Rif, andthat Rif exerts its effect allosterically by decreasing the

affinity of the RNAP for short RNA transcripts (Schulz the nucleotides further upstream (23 to 25) severelyclash with Rif. At the same time, Rif does not interfereand Zillig, 1981).

The Rif-RNAP crystal structure explains the results with the DNA (gray). Thus, the structure, in combinationwith the ternary complex model, explains the biochemi-described above and strongly supports the simple steric

block mechanism (McClure and Cech, 1978). Rif directly cal data on the mechanism of Rif inhibition, providesstrong support for the proposal that Rif sterically blocksabuts the base of a loop that comprises the C-terminal

part of b conserved region D (residues 443–451, shaded the path of the elongating RNA transcript at the 59 end,and indicates that the blockage is a direct consequencered in Figure 5; bD loop II in Korzheva et al. [2000]), and

a cluster of RifR mutants, Rif cluster I (Figure 1), flanks of Rif binding in its site. The model even suggests whytranscripts initiated with nucleoside triphosphates arethis region. Modeling suggests that this loop, which con-

tains several nearly universally conserved residues, par- blocked after the first phosphodiester bond, while tran-scripts initiated with nucleoside di- or monophosphatesticipates in forming the binding site for the base pair at

11 in the transcription complex (Korzheva et al., 2000), are blocked after the second phosphodiester bond. Inthe model, the nucleoside monophosphate in the tran-so effects of Rif on the Km for the initiating substrate

are not surprising. However, Rif does not directly con- script at the 22 position clashes only slightly with Rif,while the presence of a 59 triphosphate at the 22 posi-tact the end of this loop. In addition, conformational

changes of the protein in this region are not indicated tion would extend into Rif. The confluence of the struc-tural and biochemical data also lends support to thefrom the structural data, consistent with the observation

that the effect of Rif on this region is small. ternary complex model of Korzheva et al. (2000).Core RNAP can bind a preformed “minimal nucleicThe principal effect of Rif is seen in the context of a

model of the transcriptionally active ternary complex acid scaffold” of RNA/DNA oligonucleotides (Figure 6b,top) to yield functional ternary elongation complexes(Korzheva et al., 2000) containing RNAP, DNA template,

and RNA transcript (Figure 6). In this figure, only the (Korzheva et al., 2000). We performed order of additionexperiments using this system in order to assessRNAP active site Mg21 and the 9 bp RNA/DNA hybrid

(from 11 to 27) from the ternary complex model are whether Rif and RNA binding were competitive (Figure6b). The DNA component of the scaffold was annealedshown. The rest of the RNAP and nucleic acids are

omitted for clarity. Also shown is the atomic model of with varying lengths of RNA transcript, and the effectof Rif, added before or after the oligonucleotides, onRif as it would be positioned in its binding site on the

b subunit. the sequence-dependent extension of RNA by one nu-cleotide (radioactively labeled CTP) was assayed atIt can be seen that the two substrate nucleotides,

at 11 (green) and 21, are not directly affected by the room temperature. In the case of E. coli core RNAP inthe absence of Rif, the RNA transcript was extendedpresence of Rif, so that RNAP can bind and catalyze

the formation of a phosphodiester bond between the with nearly equal efficiency regardless of its lengthwithin a range of 3–7 nt (Figure 6b, lanes 11–15). Whentwo substrates in the presence of the antibiotic. With a

Cell908

Figure 5. Rif Binding Pocket and RifR Mutants

Stereo views of the Taq RNAP Rif binding pocket complexed with Rif. The view (the same in [a] and [b]), rotated approximately 1808 aboutthe horizontal axis from the view of Figure 4a, is as if one was in the middle of the main RNAP channel in Figure 3b (between b and b9, lookingup towards the Rif, with the b subunit behind).(a) The backbone of the b subunit is shown as a cyan ribbon, but with a highly conserved segment of region D (443–451, see text) coloredred. Side chains (and backbone atoms of F394) of residues where substitutions confer RifR (see Figure 1) are shown. Carbon atoms are orange(Rif), magenta (three residues substituted in M. tuberculosis RifR clinical isolates with high frequency, see Figure 1), yellow (other residuesthat interact directly with Rif, as in Figure 4), or green (all other RifR positions). Oxygen atoms are colored red; nitrogen atoms are blue.Generated using RIBBONS (Carson, 1991).(b) The b subunit is shown as a cyan molecular surface, with a highly conserved segment of region D colored red, and surface-exposed RifR

positions colored yellow (within 4 A of the Rif) or green. Generated using GRASP (Nicholls et al., 1991).

Rif was added prior to the nucleotide scaffold, the RNAP sion of the shortest transcripts was barely detectable,suggesting that, unlike E. coli RNAP, Taq core RNAPwas unable to extend any of the RNA oligos, regardless

of length (lanes 1–5), indicating that Rif occupied its site does not bind and stabilize the short, intrinsically unsta-ble RNA/DNA hybrids. In the presence of Rif, a general-and blocked the extension and/or binding of all of the

transcripts. When the scaffold was added prior to Rif ized inhibition of transcript extension was observed re-gardless of the order of addition or of the transcriptaddition, Rif was able to occupy its site and block the

extension of the 3 nt transcript (lane 6), but had no effect length (lanes 16–25). We explain these results by thelow binding affinity of Taq core RNAP for both Rif andon the extension of the longer transcripts (lanes 7–10),

presumably because Rif could not access its binding for short RNA transcripts compared with E. coli coreRNAP. The low affinities imply fast off rates, which wouldsite blocked by the longer RNA transcripts (Figure 6a).

These results are consistent with the early data that allow equilibrium to be established between the Rif andscaffold binding during the time of the assay.Rif inhibits the RNA extension from 2 to 3 nt if the 59

nucleoside is tri-phosphorylated, but inhibits extensionfrom 3 to 4 nt if the 59 nucleoside is mono- or di-phos- Discussionphorylated (McClure and Cech, 1978) since the syntheticRNA oligos lack 59 phosphates. In summary, we have shown that, although Taq RNAP

is relatively insensitive to Rif, at sufficiently high concen-Similar experiments were performed with Taq coreRNAP (Figure 6b, lanes 16–30). In the absence of Rif, trations, the antibiotic binds and inhibits the enzyme.

Inhibition of Taq RNAP occurs through the same bio-the efficiency of transcript extension was strongly de-pendent on the transcript length (lanes 26–30). Exten- chemical mechanism as E. coli RNAP, and the disposi-

Structure of the Rifampicin-RNA Polymerase Complex909

tion of the Rif site with respect to the active site isidentical to E. coli RNAP and presumably other prokary-otic RNAPs (Figure 2). We determined the 3.3 A X-raycrystal structure of Taq core RNAP complexed with Rif.The inhibitor bound with a close complementary fit in apocket between two structural domains of the RNAP bsubunit. Only small, local conformational changes ofboth the inhibitor and the protein were observed. Thebinding site is deep within the main RNAP channel, butthe closest approach of the inhibitor to the RNAP activesite Mg21 is more than 12 A (Figure 3b). The Rif bindingpocket is surrounded by the 23 known positions whereamino acid substitutions confer RifR (Figure 5). Twelveof these residues are close enough to interact directlywith the Rif (Figure 4). Predominant are van der Waalsinteractions with hydrophobic side chains near the nap-thol ring of Rif, and potential hydrogen bond interactionswith five polar groups of Rif (two on the napthol ringand three on the ansa bridge), four of which have beenshown to be essential for Rif activity. The remaining RifR

mutants are one layer removed from the Rif itself, andare likely to affect Rif binding through small structuraldistortions of the Rif binding pocket.

The structure explains the effects of Rif on RNAPfunction determined from detailed biochemical and ki-netic studies. In combination with a model of the ternarytranscription complex, the structure strongly suggeststhat the predominant effect of Rif is to directly block thepath of the elongating RNA transcript at the 59 end whenthe transcript becomes either 2 or 3 nt in length, de-pending on the 59 phosphorylation state of the 59 nucleo-tide (Figure 6).

In this view, Rif binds its site in the RNAP holoenzymeeither before or after the binding of the DNA templateand formation of the open complex, functions of RNAPthat are not affected by the presence of Rif. Next, thetwo nucleotide substrates bind their sites in the RNAPactive site. The initiating nucleotide substrate binds theRNAP i-site with a small, approximately 2-fold increasein the apparent Km due to the presence of Rif, while thesecond nucleotide binds in the i11 site with little effectby Rif. More or less normally, the RNAP then catalyzesthe formation of a phosphodiester bond between thetwo nucleotides. If the initiating nucleoside bears a 59triphosphate, the subsequent translocation of the RNAPattempts to move the 2 nt RNA transcript upstream suchthat the i11 nucleotide occupies the i-site (21 position),

Figure 6. Mechanism of RNAP Inhibition by Rifand the i-site nucleotide moves into the 22 position

(a) The RNAP active site Mg21 (magenta sphere) and the 9 bp RNA/ (Figure 6a). The movement of the 59 nucleotide into theDNA hybrid (from 11 to 28) from a model of the ternary elongation

22 position, however, results in a severe steric clashcomplex (Korzheva et al., 2000) are shown. The RNAP itself andwith the Rif. The molecular details of ensuing events arethe rest of the nucleic acids are omitted for clarity. The incoming

nucleotide substrate at the 11 position is colored green, the 21 unclear, but in the end, the RNAP remains at the sameand 22 positions, which can be accommodated in the presence of template position, the 2 nt transcript is released, and theRif, are colored yellow. The RNA further upstream (23 to 28), whichcannot be accommodated in the presence of Rif, is colored pink.The template strand of the DNA is colored gray. Also shown is a

of RNA (“X 5”) and analyzed on a 23% polyacrylamide gel. LanesCPK representation of Rif as it would be positioned in its binding1–10 and 16–25 demonstrate the effect of Rif inhibition of transcrip-site on the b subunit (carbon atoms, orange; oxygen, red; nitrogen,tion when it was bound by RNAP either before (lanes 1–5 and 16–20)blue). The Rif is partially transparent, illustrating the RNA nucleotidesor after (lanes 6–10 and lanes 21–25) addition of the scaffold. Lanesat 23 to 25 that sterically clash. Generated using GRASP (Nicholls11–15 and 26–30 show elongation of the same scaffolds in theet al., 1991).absence of Rif. The RNA with the critical length of 3 nt, which cannot(b) The structure of the minimal scaffold systems with RNA lengthsbe elongated by E. coli RNAP in the presence of Rif regardless offrom 3–7 nt (labeled above the RNA chain; Korzheva et al., 2000).the order of Rif and scaffold addition (lanes 1 and 6), is colored red.The results are presented below as autoradiographs of the radioac-The RNAs of 4–7 nt (colored green) were extended by E. coli RNAPtive RNAs produced by E. coli (lanes 1–15) or Taq (lanes 16–30) corewhen added before Rif (lanes 6–10).RNAPs transcribing the minimal scaffolds with the indicated lengths

Cell910

futile cycle begins again. If the 59 nucleoside contains a because of the apparently small functional penalties ofdi- or a monophosphate at its 59 end (or if it’s unphos- mutating this region of the RNAP, and the variety ofphorylated), then after the synthesis of the first phospho- amino acid positions and mutations that result in RifR

diester bond, the RNAP can translocate normally and (Figure 1). Somewhat encouraging, however, are thethe steric clash of the transcript with Rif occurs during findings from clinical isolates of RifR M. tuberculosis.the translocation of the 3 nt transcript following the Although the RifR mutations are spread over 15 positionssynthesis of the second phosphodiester bond. of rpoB, 77% of all the mutations isolated involved sub-

The relative insensitivity of Taq RNAP to Rif is likely stitutions at one of only two positions, correspondingdue to amino acid substitutions in Taq RNAP compared to Taq 406 and 411, and an additional position (Taq 396)with other, more Rif-sensitive RNAPs. The 12 residues accounts for a combined 86% of all the mutants.close enough to interact directly with Rif are identical An important conclusion from this study emerges frombetween E. coli, Taq, and M. tuberculosis (marked yellow the inhibition mechanism of Rif, a simple steric block ofin Figure 1). Among the 11 positions that do not directly transcription elongation. Thus, the powerful effects ofinteract with Rif but likely affect Rif binding indirectly, Rif do not stem from the details of its chemical structure,5 are substituted in Taq RNAP (387, 395, 398, 453, and and do not involve interference with the catalytic activity566; Figure 1). Although the effect of these residues on of the RNAP, for instance by mimicking substrates orthe structure of RNAP is difficult to assess with only one transition states of the polymerization reaction. Such anavailable structure, we can venture some suppositions. inhibitor would act on features that are highly conservedThree of these positions, 387, 398, and 453, contain between prokaryotes and eukaryotes, rendering it use-amino acids that are not dramatically different in overall less as an antimicrobial agent. Rather, the effects of Rifsize from their E. coli and M. tuberculosis counterparts depend only on its ability to bind tightly to a relativelyand we predict that these residues are not the origin of nonconserved part of the structure, disrupting a criticalthe Taq RNAP insensitivity to Rif. Position 566 is highly RNAP function by virtue of its presence. Decades ofconserved among all RNAPs as either Lys or Arg (the functional studies (Chamberlin, 1993; Mustaev et al.,homologous position is Arg in both E. coli and M. tuber- 1997; Korzheva et al., 1998; Nudler, 1999), and moreculosis) but is Thr in Taq RNAP. This substitution is recent structural evidence (Mooney and Landick, 1999;unlikely to be the main determinant of the Taq RNAP Zhang et al., 1999; Cramer et al., 2000; Korzheva et al.,Rif insensitivity, however, since Minakhin et al. (2001b) 2000), indicate that cellular RNAPs operate as complexmutated Taq Thr-566 to Arg, but this had little effect on molecular machines, with extensive interactions withthe RifR of the enzyme assayed at 458C. This leaves the template DNA, product RNA (Korzheva et al., 2000),position 395, which is highly conserved as a hydropho- and other regulatory molecules. It seems likely that manybic residue among all bacterial RNAPs. In E. coli and distinct sites exist where the tight binding of a smallM. tuberculosis, this position is a Met, but in Taq, it is molecule would disrupt critical features of the functionala Lys. Taq Lys-395 appears to participate in buried salt mechanism.bridges with Asp-124 and Asp-133 that may contributeto thermostability of the protein. This nonconservative Experimental Proceduressubstitution (Lys for Met) could affect the local path of

Purification and Crystallizationthe polypeptide backbone, and is immediately adjacentNative Taq core RNAP was purified and crystallized as describedto Phe-394, the backbone amide and carboxyl of whichpreviously (Zhang et al., 1999). The crystals were then soaked inappear to be involved in important interactions with thestabilization solution (2 M (NH4)2SO4, 0.1 M Tris-HCl, pH 8.0, and 20

Rif (Figure 4). mM MgCl2) with 0.1 mM Rif for at least 12 hr. The crystals wereAll but one of the residues that are close enough to then prepared for cryo-crystallography by soaking in stabilization

Rif to participate in direct interactions are known to solution containing 50% (w/v) sucrose for 30 min before flash freez-ing in liquid nitrogen. Diffraction data was collected at the APSmutate to strong RifR (Figure 4). However, additionalbeamline SBC 19ID using 0.38 oscillations, and processed usingresidues could be important for the formation of the RifDENZO and SCALEPACK (Otwinowski, 1991).binding pocket, but not revealed as RifR mutants if they

are necessary for basic RNAP function. As mentioned Structure Determinationabove, the four regions of the b subunit that harbor The native core RNAP structure (Zhang et al., 1999) was used as aRifR mutants are highly conserved among prokaryotes starting model for rigid body refinement and positional refinement(Figure 1), but the much weaker homology with archae- against the observed amplitudes from the Rif-RNAP complex crystal

(FoRif:) using CNS (Adams et al., 1997), yielding an initial R factor ofbacterial and eukaryotic RNAPs, combined with the fact

0.354 (Rfree 5 0.41, where the identical reflections was set aside asthat so many RifR mutations have been discovered, indi-for the Rfree determination of the native structure) for data from 100-cate that these regions are not critical to RNAP function3.2 A resolution. An initial Fourier difference map, calculated using

in vivo. Nevertheless, some RifR mutations do have pro- |FoRif 2 Fo

nat| amplitude coefficients and using phases calculated fromfound functional effects (Landick et al., 1990; Jin and the native core RNAP structure (φnat), clearly revealed density forGross, 1991), and E. coli strains with RifR RNAP have the Rif molecule (Figure 3a). Multiple rounds of manual rebuildingbeen shown to be at a competitive disadvantage to wt against (2|Fo| 2 |Fc|) maps using O (Jones et al., 1991), and refinement

using CNS (Adams et al., 1997), resulted in the current model (TableE. coli in the absence of Rif (Jin and Gross, 1989).1). At later stages of the refinement, The Rif X-ray crystal structureThe clinical success of Rif proves that the bacterial(Brufani et al., 1974) was easily placed into the difference density.RNAP is an excellent target for antimicrobials. The struc-Included in the model is the recently determined sequence of the

ture and available genetic and biochemical data suggest Taq v subunit (Minakhin et al., 2001a), modeled earlier as a polyala-that the design of modified versions of Rif to overcome nine chain (Zhang et al., 1999). Still missing from the model is athe effects of RifR mutations, while perhaps leading to 300 amino acid, nonconserved domain inserted between conserved

regions A and B of the b9 subunit (Zhang et al., 1999).incremental improvements, may be futile in the long run

Structure of the Rifampicin-RNA Polymerase Complex911

Assays simulated annealing refinement. Proc. Natl. Acad. Sci. USA 94, 5018–5023.Taq cells were tested for sensitivity to Rif on solid media. Plates

containing 3% bactoagar and 1/5 dilution of Luria broth were poured Allison, L.A., Moyle, M., Shales, M., and Ingles, C.J. (1985). Extensivewith and without 50 mg/ml of Rif. Cells from frozen stock were then homology among the largest subunits of eukaryotic and prokaryoticstreaked onto plates and incubated at 658C for 2 days and assessed RNA polymerases. Cell 42, 599–610.for growth.

Archambault, J., and Friesen, J.D. (1993). Genetics of RNA polymer-The transcription assay comparing Rif inhibition of E. coli and Taq

ases I, II, and III. Microbiol. Rev. 57, 703–724.RNAPs (Figure 2a) was performed as described (Nudler et al., 1994).

Arora, S.K. (1981). Structural investigations of mode of action ofBriefly, 0.1 pmol of purified Taq core RNAP (Zhang et al., 1999) wasdrugs III. Structure of rifamycin S iminomethyl ether. Acta crys-incubated with Taq sA (Minakhin et al., 2001b) in 20 ml of transcriptiontallogr. B37, 152–157.buffer (40 mM Tris-HCl, pH 7.9, 40 mM KCl, 5 mM MgCl2) for 15 min at

378C to form holoenzyme. Rif was added to the final concentrations Arora, S.K. (1983). Correlation of structure and activity in ansamy-indicated in Figure 2a and incubated another 5 min at 378C, followed cins: molecular structure of sodium rifamycin SV. Mol. Pharmacol.by addition of 0.15 pmol of T7A1 promoter fragment and incubation 23, 133–140.for 5 min at 378C. RNA synthesis was initiated by the addition of Arora, S.K. (1985). Correlation of structure and activity in ansamy-CpA primer (100 mM), NTPs (25 mM each), and a-[32P]UTP (0.3 mM), cins: structure, conformation, and interactions of antibiotics rifamy-and the reaction was stopped after incubation for 10 min at 378C. cin S. J. Med. Chem. 28, 1099–1102.The assay for E. coli RNAP holoenzyme was the same except the

Arora, S.K., and Main, P. (1984). Correlation of structure and activityCpA primer was added to a concentration of 10 mM. Radioactivein ansamycins: moleculr structure of cyclized rifamycin SV. J. Anti-RNA products were analyzed on a 15% polyacrylamide sequenc-biot. 37, 178–181.ing gel.Brufani, M., Cerrini, S., Fidelli, W., and Vaciago, A. (1974). Rifamy-Assays for extension of the Rif-nucleotide compounds (Figure 2b)cins, an insight into biological activity based on structural investiga-were carried out as described (Mustaev et al., 1994) with minortions. J. Mol. Biol. 87, 409–435.modifications. After binary complex formation, transcription reac-

tions were started by the addition 10 mM Rif-(CH2)n-A compound, Carson, M. (1991). RIBBONS 2.0. J. App. Crystallogr. 24, 958–961.with the “n” indicated in Figure 2b, and a-[32P]UTP (0.3 mM). The Chamberlin, M.J. (1993). New models for the mechanism of tran-reactions were incubated for 2 min at room temperature for E. coli scription elongation and its regulation. Harvey Lect. 88, 1–21.RNAP and 3 min at 558C for Taq. Under these conditions, the reaction

Cramer, P., Bushnell, D.A., Fu, J., Gnatt, A.L., Maier-Davis, B.,was not complete, and the yield of the Rif-(CH2)n-ApU depended onThompson, N.E., Burgess, R.R., Edwards, A.M., David, P.R., andthe linker length. Radioactive RNA products were analyzed on aKornberg, R.D. (2000). Architecture of RNA polymerase II and impli-23% polyacrylamide sequencing gel.cations for the transcription mechanism. Science 288, 640–649.Transcription reactions on the minimal scaffold system shownDrancourt, M., and Raoult, D. (1999). Characterization of mutations(Figure 6b) were performed as described (Korzheva et al., 2000) within the rpoB gene in naturally rifampin-resistant Rickettsia species.minor modifications. The RNA and DNA components of the scaffoldAntimicrob. Agents and Chemotherapeutics 43, 2400–2403.(100 pmol of each) were mixed in 100 ml of transcription buffer at

458C and the mixture was allowed to cool to room temperature over Ezekiel, D.H., and Hutchins, J.E. (1968). Mutations affecting RNAP30 min. RNAP/scaffold complexes were formed by incubation of associated with rifampicin resistance in Escherichia coli. Naturethe annealed scaffold (10 pmol) with a molar equivalent of core London 220, 276–277.RNAP (either E. coli or Taq) preincubated with Rif (100 mM for E.

Hartmann, G., Honikel, K.O., Knusel, F., and Nuesch, J. (1967). Thecoli, 200 mM for Taq) for 10 min, to form the RNAP/scaffold complex.

specific inhibition of the DNA-directed RNA synthesis by rifamycin.Extension of the RNA oligonucleotide was assayed by the addition

Biochim. Biophys. Acta 145, 843–844.of a-[32P]CTP (0.3 mM) and 5 min incubation at room temperature.

Heep, M., Beck, D., Bayerdorffer, E., and Lehn, N. (1999). RifampinIn Figure 6b, lanes 1–5 and 16–20, RNAP was preincubated with Rifand rifabutin resistance mechanism in Helicobacter pylori. Antimi-(100 mM for E. coli RNAP, 200 mM for Taq) for 10 min. In lanes 6–10crob. Agents and Chemotherapeutics 43, 1497–1499.and 21–25, the RNAP/scaffold complexes formed in the absence of

Rif were incubated with Rif (concentrations as above) for 10 min. Heep, M., Rieger, U., Beck, D., and Lehn, N. (2000). Mutations inthe beginning of the rpoB gene can induce resistance to rifamycinsFinally, in lanes 11–15 and 26–30, the RNAP or RNAP/scaffold com-

plex was not exposed to Rif. Radioactive RNA products were ana- in both Helicobacter pylori and Mycobacterium tuberculosis. Antimi-crob. Agents and Chemotherapeutics 44, 1075–1077.lyzed on a 23% polyacrylamide sequencing gel.

Heil, A., and Zillig, W. (1970). Reconstitution of bacterial DNA-depen-dent RNA polymerase from isolated subunits as a tool for the eluci-Acknowledgmentsdation of the role of the subunits in transcription. FEBS Lett. 11,165–168.We are indebted to A. Joachimiak, S. L. Ginell, and F. J. Rotella at

the Advanced Photon Source Structural Biology Center for support Hinkle, D.C., Mangel, W.F., and Chamberlin, M.J. (1972). Studies ofduring data collection. Use of the Argonne National Laboratory the binding of Escherichia coli RNA polymerase to DNA. IV. TheStructural Biology Center beamlines at the Advanced Photon Source effect of rifampicin on binding and on RNA chain initiation. J. Mol.was supported by the U. S. Department of Energy, Office of Biologi- Biol. 70, 209–220.cal and Environmental Research, under Contract No. W-31-109- Honore, N., Bergh, S., Chanteau, S., Doucet-Populaire, F., Eiglmeier,ENG-38. We thank V. Nikiforov and J. McKinney for invaluable dis- K., Garnier, T., Georges, C., Launois, P., Limpaiboon, T., Newton,cussions. E. C. was supported by a Kluge postdoctoral fellowship S., et al. (1993). Nucleotide sequence of the first cosmid from theand a National Research Service Award (NIH GM20470). K. M. was Mycobacterium leprae genome project: structure and function ofsupported by a Human Frontiers Sciences Program postdoctoral the Rif-Str regions. Mol. Microbiol. 7, 207–214.fellowship and a Norman and Rosita Winston postdoctoral fellow-

Jin, D.J., and Gross, C.A. (1988). Mapping and sequencing of muta-ship. This work was supported by NIH grants GM49242 andtions in the Escherichia coli rpoB gene that lead to rifampicin resis-GM30717 to A. G., and GM53759 and GM61898 to S. A. D.tance. J. Mol. Biol. 202, 45–58.

Jin, D.J., and Gross, C.A. (1989). Characterization of the pleiotropicReceived December 13, 2000; revised January 31, 2001. phenotypes of rifampin-resistant rpoB mutants of Escherichia coli.

J. Bacteriol. 171, 5229–5231.

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