sample is physically larger, any mechanical errorsin post-expansion sectioning, or stage drift, aredivided by the expansion factor.The performance of ExM suggests that de-

spite statistical fluctuations in polymer chainlength at the molecular scale, at the nanoscaledistances here examined these fluctuations av-erage out, yielding isotropy. Estimates of meshsize for comparable gels suggest that the dis-tance between nearest-neighbor polymer chainsare in the ~1 to 2 nm range (17, 18). By tuningthematerial properties of the ExM polymer, suchas the density of cross-links, yet higher effectiveresolutions may be possible.

REFERENCES AND NOTES

1. T. Tanaka et al., Phys. Rev. Lett. 45, 1636–1639 (1980).2. I. Ohmine, J. Chem. Phys. 77, 5725 (1982).3. F. L. Buchholz, Superabsorbent Polymers 573, 27–38 (1994).4. B. Huang, S. A. Jones, B. Brandenburg, X. Zhuang, Nat.

Methods 5, 1047–1052 (2008).5. E. H. Rego et al., Proc. Natl. Acad. Sci. U.S.A. 109, E135–E143

(2012).6. M. Bates, B. Huang, G. T. Dempsey, X. Zhuang, Science 317,

1749–1753 (2007).7. N. Olivier, D. Keller, P. Gönczy, S. Manley, PLOS One 8, (2013).8. R. W. Cole, T. Jinadasa, C. M. Brown, Nat. Protoc. 6, 1929–1941

(2011).9. G. Feng et al., Neuron 28, 41–51 (2000).10. A. Dani, B. Huang, J. Bergan, C. Dulac, X. Zhuang, Neuron 68,

843–856 (2010).11. A. Rollenhagen, J. H. R. Lübke, Front. Synaptic Neurosci. 2,

2 (2010).12. G. R. Newman, B. Jasani, E. D. Williams, Histochem. J. 15,

543–555 (1983).13. K. D. Micheva, S. J. Smith, Neuron 55, 25–36 (2007).14. B.-C. Chen et al., Science 346, 1257998 (2014).15. J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, E. H. K. Stelzer,

Science 305, 1007–1009 (2004).16. J. H. Lee et al., Science 343, 1360–1363 (2014).17. A. M. Hecht, R. Duplessix, E. Geissler, Macromolecules 18,

2167–2173 (1985).18. D. Calvet, J. Y. Wong, S. Giasson, Macromolecules 37,

7762–7771 (2004).

ACKNOWLEDGMENTS

E.S.B. was funded by NIH Director’s Pioneer Award 1DP1NS087724and NIH Director’s Transformative Research Award 1R01MH103910-01,the New York Stem Cell Foundation-Robertson Investigator Award, theMIT Center for Brains, Minds, and Machines NSF CCF-1231216,Jeremy and Joyce Wertheimer, Google, NSF CAREER Award CBET1053233, the MIT Synthetic Intelligence Project, the MIT MediaLab, the MIT McGovern Institute, and the MIT NeurotechnologyFund. F.C. was funded by an NSF Graduate Fellowship. P.W.T.was funded by a Fannie and John Hertz Graduate Fellowship. Confocalimaging was performed in the W. M. Keck Facility for BiologicalImaging at the Whitehead Institute for Biomedical Research.Deltavision OMX SR-SIM imaging was performed at the Koch InstituteSwanson Biotechnology Center imaging core. We acknowledgeW. Salmon and E. Vasile for assistance with confocal and SR-SIMimaging. We acknowledge N. Pak for assistance with perfusions.We also acknowledge, for helpful discussions, B. Chow,A. Marblestone, G. Church, P. So, S. Manalis, J.-B. Chang,J. Enriquez, I. Gupta, M. Kardar, and A. Wissner-Gross. Theauthors have applied for a patent on the technology, assignedto MIT (U.S. Provisional Application 61943035). The order ofco–first author names was determined by a coin toss. Theimaging and other data reported in the paper are hosted by MIT(http://expansionmicroscopy.org/rawdataScience2014).

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/347/6221/543/suppl/DC1Materials and MethodsFigs. S1 to S5Tables S1 to S4References (19–28)Movies S1 to S3

18 August 2014; accepted 26 November 201410.1126/science.1260088

MITOCHONDRIAL BIOLOGY

Replication-transcription switch inhuman mitochondriaKaren Agaronyan, Yaroslav I. Morozov, Michael Anikin, Dmitry Temiakov*

Coordinated replication and expression of themitochondrial genome is critical formetabolicallyactive cells during various stages of development. However, it is not known whether replicationand transcription can occur simultaneously without interfering with each other and whethermitochondrial DNAcopynumbercanbe regulatedby the transcriptionmachinery.We found thatinteraction of human transcription elongation factor TEFM with mitochondrial RNA polymeraseand nascent transcript prevents the generation of replication primers and increasestranscription processivity and thereby serves as a molecular switch between replication andtranscription, which appear to be mutually exclusive processes in mitochondria. TEFM mayallow mitochondria to increase transcription rates and, as a consequence, respiration andadenosine triphosphate production without the need to replicate mitochondrial DNA, as hasbeen observed during spermatogenesis and the early stages of embryogenesis.

The maternally inherited circular mitochon-drial DNA (mtDNA) encodes subunits ofcomplexes of the oxidative phosphoryla-tion chain, aswell as transfer RNAs (tRNAs)and ribosomal RNAs (1, 2). Transcription of

humanmtDNA is directed by two promoters, theLSP (light-strand promoter) and the HSP (heavy-strand promoter) located in opposing mtDNAstrands, which results in two almost-genome-sized polycistronic transcripts that undergo ex-tensive processing before polyadenylation andtranslation (3, 4). Note that transcription ter-minates prematurely about 120 base pairs (bp)downstream of LSP at a vertebrate-conservedG-rich region, called conserved sequence blockII (CSBII), as a result of formation of a hybridG-quadruplex between nascent RNA and thenontemplate strand of DNA (5–7). This termi-nation event occurs near the origin of replicationof the heavy strand (oriH) (8) and generates areplication primer. According to the asymmetricmodel (9), replication then proceeds through abouttwo-thirds of themtDNA, until the oriL sequencein the opposing strand becomes single strandedand forms a hairpin structure. The oriL hairpin isthen recognized bymitochondrial RNApolymerase(mtRNAP), which primes replication of the lightstrand (10). Because replication of mtDNA coin-cideswith transcription in timeand space, collisionsbetween transcription and replication machine-ries are inevitable and, similarly to bacterial andeukaryotic systems, likely have detrimental effectson mtDNA gene expression (11).We analyzed the effects of a mitochondrial

transcription elongation factor, TEFM, recentlydescribed by Minczuk and colleagues (12), ontranscription of mtDNA. This protein was pulleddown from mitochondrial lysates via mtRNAPand was found to stimulate nonspecific transcrip-tion on promoterless DNA; however, its effecton promoter-driven transcription had not been

determined (12). We found that in the presenceof TEFM,mtRNAP efficiently transcribes throughCSBII (Fig. 1, A and B). Thus, TEFM acts as a fac-tor that prevents termination at CSBII and syn-thesis of a primer for mtDNA polymerase. Weidentified the exact location of the terminationpoint in CSBII (fig. S1). MtRNAP terminates atthe end of a U6 sequence (positions 287 to 283inmtDNA), 16 to 18 nucleotides (nt) downstreamof the G-quadruplex (Fig. 1A). At this point, the9-bp RNA-DNA hybrid in the elongation com-plex (EC) is extremely weak, as it is composed ofonly A-U and T-A pairs. This is reminiscent ofintrinsic termination signals in prokaryotes—where the formation of anRNAhairpin is thoughtto disrupt the upstream region of the RNA-DNAhybrid—and is followed by the run of six to eighturidine 5′-monophosphate residues that furtherdestabilizes the complex (5, 13).Human mtDNA is highly polymorphic in the

CSBII region; coincidently, the reference mito-chondrial genome (Cambridge) contains a rarepolymorphism in the G-quadruplex—namely,G5AG7—whereas the majority of mtDNAs fromvarious haplogroups have two additional G resi-dues (G6AG8) (14).We found that the terminationefficiency of mtRNAP was substantially lower atG5AG7-CSBII (Fig. 1C), which suggested an effectof G run length on quadruplex formation andunderscored the importance of further studies ofvarious polymorphisms in this region.In considering a putativemechanism of TEFM

antitermination activity, we investigated wheth-er it can interact with the nascent transcript and,thus, interfere with the formation of the quad-ruplex structure. We assembled ECs on a nucleicacid scaffold containing a photoreactive analogof uridine, 4-thio-uridine, 13 nt downstream fromthe 3′ end of RNA, andwalkedmtRNAP along thetemplate by incorporation of appropriate substratenucleoside triphosphates (NTPs) (Fig. 2A). We ob-served efficient cross-linking between TEFM andRNAwhen the photoreactive base was 15 to 16 bpaway from the 3′ end of RNA. Additionally, usinga templateDNAcontaining the LSP promoter and

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Department of Cell Biology, School of Osteopathic Medicine,Rowan University, 2 Medical Center Drive, Stratford, NJ08084, USA.*Corresponding author. E-mail:temiakdm@rowan.edu

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CSBII, we probed interactions of TEFMwith RNAhaving a photoreactive probe 6-thio guanosine-5′-monophosphate (6-thio GMP) 9 to 16 or 18 to25 nt away from the 3′ end of RNA (fig. S2). Wefound that TEFM efficiently cross-linked to theG-quadruplex region of RNA, which further con-firmed its interactions with the nascent transcript(fig. S2).The catalytic domains of mtRNAP and the

single-subunit RNAP from bacteriophage T7 sharehigh sequence and structural homology (15).When we compared the structural organizationof humanmtRNAP EC (15) and T7 RNAPEC (16),one striking difference became apparent—theformer polymerasemakes very few contacts withthe downstreamDNAduplex (fig. S3). In contrast,

in T7 RNAP EC, the N terminus of the polymerasemakes extensive interactionswith the entire down-stream duplex. These extensive interactions withthe downstream DNA may explain the high pro-cessivity of T7 RNAP (16). Because TEFM bindsthe C terminus of mtRNAP (12), we hypothesizedthat it may also bind the downstream DNA tocompensate for the lack of mtRNAP-DNA inter-actions. To evaluate this, we incorporated 4-thio-2′-deoxythymidine-5′-monophosphate (4-thio dTMP)into the DNA template in the downstream regionand assembled the EC using RNA-DNA scaffolds.We found that TEFM efficiently cross-linked tothe +7 and +8 bases in the DNA template strand(Fig. 2B and fig. S4A). The DNA-TEFM cross-linking was species-specific, as no efficient cross-

linking was detected when yeast mtRNAP wasused (fig. S4B).The binding site of TEFMonmtRNAPhas been

previously identified in the C-terminal region, be-tween residues 768 and 1230 (12). Thesedata, takentogether with the RNA and DNA cross-linkingdata, suggest that TEFM likely binds to the palmsubdomain of mtRNAP in proximity to the PPRdomain and interacts with the emerging RNAtranscript (Fig. 2C). It is therefore likely thatTEFM antitermination activity is due to directinterference with the formation of the bulkyG-quadruplex structure between the palm sub-domain and the PPR domain of mtRNAP or dueto the allosteric effect of quadruplex formationon complex stability. Similar mechanisms of

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Fig. 1. TEFM prevents terminationat CSBII. (A) Schematic illustrationof the human mtDNA regiondownstream of the LSP promoter.The numbers below the schemecorrespond to the reference humanmtDNA; the transcribed sequenceof CSBII is shown at the top.(B) MtRNAP does not terminate atCSBII in the presence of TEFM.Termination efficiency (%) is indi-cated below the panel. (C) A rarepolymorphism in a reference mtDNAresults in a decreased efficiency oftranscription termination at CSBII.

LSP CSB III CSB II CSB I

408

363 315 235345 283 212

terminationregion (283-287)

G-quadruplex region(G6AG8)

Time, min

- TEFM + TEFM20 30 40 50 60 20 30 40 50 60

Run-off200 nt

replication primer(termination at CSBII)

~120 nt

1 2 3 4 5 6 7 8 9 1095% 5%

G6AG8 G5AG7reference genome,rare polymorphism

(most genomes)

Time, min 20 30 40 50 60 20 30 40 50 60

90% 60%

Run-off,200 nt

replication primer(termination at CSBII)

~120 nt

1 2 3 4 5 6 7 8 9 10

-13 -14 -15 -16

1 2 3 4

A,C

A,C

,3'd

G

Ano

ne

NTPs

mtRNAP/RNA

TEFM/RNA

16 nt15 nt14 nt

13 nt

RN

A

Thumb

Fingers

PPR

region of interactionwith TEFM

N-terminal domain

Palm

RNAbase at -18

downstreamDNA

+7+8

1 2 3 4 5

C,3

'dA

C,A

,3'd

G

C,A

3'd

C

no

ne

NTPs

+8 +7 +6 +5 +4

mtRNAP/DNA

TEFM/DNA

16 nt

15 nt

14 nt

13 nt

12 nt

RN

A

35 -13

Fig. 2. Interactions of TEFM with the components of the elon-gation complex. (A) TEFM interacts with the RNA in a scaffoldEC. (Top) Cross-linking of labeled RNA to the proteins indicated;(bottom) RNA extension. (B) TEFM interacts with the downstreamDNA. (C) Putative region of interaction with TEFM. Structure of

humanmtRNAP EC (surface representation, PDB ID 4BOC) is shown with the major domains indicated (fingers, pink; palm, light green; N-terminal domain, darkgreen; thumb, orange; DNA template strand, blue; nontemplate strand, cyan; and RNA, red). The 9-nt-long RNA in the EC is extended by 9 nt to model thetrajectory of the nascent RNA.

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antitermination have been suggested for lambdaphage antiterminators Q and N and a number ofbacterial antiterminators that were shown to inter-act with both RNAP and its nascent transcript thatwould otherwise form a hairpin structure (17, 18).Because TEFM interacts with the downstream

DNA (whichmay stabilize the EC), we decided toinvestigate its effect on mtRNAP processivity.We found that the efficiency of runoff synthesisby mtRNAP was low if the size of the productexceeded 500 nt (Fig. 3, A and B). Indeed, theprocessivity of mtRNAP was substantially lowerthan that of a related T7 RNAP (which is known totranscribe large fragments of DNA) andwas appar-ently not sufficient to transcribe a 16,000-bp-longmtDNA. In the presence of TEFM, the efficiencyof synthesis of long RNA was significantly in-creased (Fig. 3, A and B), which suggested thatTEFM indeed acts as a processivity factor, as wasoriginally proposed (12).We next probed if TEFM contributes to the

stability of the elongating mtRNAP and, as a con-sequence, to its antitermination activity. To dem-onstrate this, we halted the EC 35 bp downstreamof the promoter start site by omitting one of thesubstrate NTPs [cytidine 5′-triphosphate (CTP)](Fig. 3C and fig. S5), in the presence or absence ofTEFM. After 10 to 40 min, the complexes werechased to allow synthesis of a runoff product.In the absence of TEFM, accumulation of a 35-oligomer RNA product predominated, with littleproduction of runoff, which indicates that thehalted EC is unstable, rapidly dissociates andreinitiates, but is inefficiently chased (Fig. 3C andfig. S5). In contrast, in the presence of TEFM, lit-tle, if any, of 35-oligomer RNA product accumu-lates, but more efficient synthesis of full-lengthproducts is observed, which indicates that, underthese conditions, the EC is stable and may sub-sequently be extended.Our in vitro data demonstrate that TEFM in-

creases the overall processivity of ECs. In theabsence of TEFM, mtRNAP is unable to effi-ciently produce long transcripts and, instead,terminates at CSBII, which primes replication ofthe heavy strand of mtDNA (Fig. 4). Consistentwith this, a knockdown of TEFM in human cellsresults in a decrease of promoter-distal RNA tran-scripts (12). We therefore propose that replicationand transcription aremutually exclusive processesin human mitochondria that allow the corre-sponding machineries to avoid the detrimentalconsequences of head-on collisions (Fig. 4). Assuch, TEFM serves as a molecular switch thatwould allow the organelles to either replicate themtDNA and regulate its copy number or to ele-vate its transcription rates. This switch can be im-portant for a number of developmental processes,such as embryogenesis and spermatogenesis,during which an active transcription of mtDNAis observedwithout replication (19–25). AlthoughTEFMmay not be the only factor affectingmtDNAcopy number (26), the proposed mechanism pro-vides a solution to these important biological pro-cesses. Further in vivo analysis will be required toaddress the question as to how this switch is regu-lated inmitochondria of activelymetabolizing cells.

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no TEFM TEFM

10 20 30 40 10 20 30 40

500 nt

HaltedEC

35 nt

Time beforechase, min

1 2 3 4 5 6 7 82% 16%

1 2 3 4 5 6 7 8

TEFMno TEFMtime, min 10 20 30 40 10 20 30 40

500 nt

TEFMno TEFM

1000 nt

1 2 3 4 5 6 7 8

time, min 10 20 30 40 10 20 30 40

100%37%

31%10%

Fig. 3. TEFM increases processivity and stability of mtRNAP EC. (A and B) TEFM increases the ECprocessivity.Transcriptionwas performed using LSP templates lackingCSBII sequence to generate 500-nt(A) or 1000-nt (B) runoff products. Efficiency of synthesis of 500 nt of RNA in the presence of TEFM istaken as 100%. (C) TEFM increases the EC stability. Complexes halted at +35 in the presence or absenceof TEFMwere incubated for the times indicated and chasedwithCTP.Numbers below indicate efficiencyofa halted EC extension (%).

Fig. 4. Model for a replication-transcription switch in mitochondria. In the absence of TEFM,mtRNAP initiates transcription at the LSP promoter but terminates at CSBII and produces a 120-ntreplication primer at the replication origin oriH.The primer is used by replisome for replication of the heavystrand ofmtDNA, duringwhich a separate initiation event involvingmtRNAPgenerates a replication primerat oriL. Because of low processivity, transcription from the HSP does not produce a full-size transcript(bottom left). When TEFM is bound (bottom right), transcription from HSP produces genome-lengthtranscripts. At the same time, LSP-driven transcription is not terminated at CSBII and proceeds to the siteof factor-dependent termination (MTERF).

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REFERENCES AND NOTES

1. M. Falkenberg, N. G. Larsson, C. M. Gustafsson, Annu. Rev.Biochem. 76, 679–699 (2007).

2. N. D. Bonawitz, D. A. Clayton, G. S. Shadel, Mol. Cell 24,813–825 (2006).

3. B. M. Hällberg, N. G. Larsson, Cell Metab. 20, 226–240(2014).

4. Y. I. Morozov et al., Nucleic Acids Res. 42, 3884–3893 (2014).5. P. H. Wanrooij et al., Nucleic Acids Res. 40, 10334–10344

(2012).6. P. H. Wanrooij, J. P. Uhler, T. Simonsson, M. Falkenberg,

C. M. Gustafsson, Proc. Natl. Acad. Sci. U.S.A. 107,16072–16077 (2010).

7. D. D. Chang, D. A. Clayton, Proc. Natl. Acad. Sci. U.S.A. 82,351–355 (1985).

8. D. Kang, K. Miyako, Y. Kai, T. Irie, K. Takeshige, J. Biol. Chem.272, 15275–15279 (1997).

9. D. A. Clayton, Cell 28, 693–705 (1982).10. J. M. Fusté et al., Mol. Cell 37, 67–78 (2010).

11. R. T. Pomerantz, M. O’Donnell, Cell Cycle 9, 2537–2543(2010).

12. M. Minczuk et al., Nucleic Acids Res. 39, 4284–4299(2011).

13. V. Epshtein, C. J. Cardinale, A. E. Ruckenstein, S. Borukhov,E. Nudler, Mol. Cell 28, 991–1001 (2007).

14. R. M. Andrews et al., Nat. Genet. 23, 147 (1999).15. K. Schwinghammer et al., Nat. Struct. Mol. Biol. 20, 1298–1303

(2013).16. T. H. Tahirov et al., Nature 420, 43–50 (2002).17. I. Gusarov, E. Nudler, Cell 107, 437–449 (2001).18. S. Shankar, A. Hatoum, J. W. Roberts, Mol. Cell 27, 914–927

(2007).19. A. Rantanen, N. G. Larsson, Hum. Reprod. 15 (suppl. 2), 86–91

(2000).20. J. St John, Biochim. Biophys. Acta 1840, 1345–1354 (2014).21. T. Wai et al., Biol. Reprod. 83, 52–62 (2010).22. P. J. Carling, L. M. Cree, P. F. Chinnery, Mitochondrion 11,

686–692 (2011).

23. J. Van Blerkom, Mitochondrion 11, 797–813 (2011).24. S. Monnot et al., Hum. Mol. Genet. 22, 1867–1872 (2013).25. D. C. Wallace, Annu. Rev. Genet. 39, 359–407 (2005).26. M. I. Ekstrand et al., Hum. Mol. Genet. 13, 935–944 (2004).

ACKNOWLEDGMENTS

We thank K. Schwinghammer for help in cloning and isolationof TEFM and W. McAllister and M. Gottesman for the criticaldiscussion and reading of the manuscript. This work wassupported in part by NIH R01GM104231.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/347/6221/548/suppl/DC1Materials and MethodsFigs. S1 to S5References

16 October 2014; accepted 23 December 201410.1126/science.aaa0986

PROTEIN STRUCTURE

Structure and activity oftryptophan-rich TSPO proteinsYouzhong Guo,1 Ravi C. Kalathur,2 Qun Liu,2,3 Brian Kloss,2 Renato Bruni,2

Christopher Ginter,2 Edda Kloppmann,2,4 Burkhard Rost,2,4 Wayne A. Hendrickson1,2,3,5*

Translocator proteins (TSPOs) bind steroids and porphyrins, and they are implicated inmany human diseases, for which they serve as biomarkers and therapeutic targets. TSPOshave tryptophan-rich sequences that are highly conserved from bacteria to mammals. Herewe report crystal structures for Bacillus cereus TSPO (BcTSPO) down to 1.7 Å resolution,including a complex with the benzodiazepine-like inhibitor PK11195. We also describeBcTSPO-mediated protoporphyrin IX (PpIX) reactions, including catalytic degradation toa previously undescribed heme derivative. We used structure-inspired mutations toinvestigate reaction mechanisms, and we showed that TSPOs from Xenopus and man havesimilar PpIX-directed activities. Although TSPOs have been regarded as transporters,the catalytic activity in PpIX degradation suggests physiological importance for TSPOs inprotection against oxidative stress.

The translocator protein (TSPO) is namedfor its putative roles in the transport ofcholesterol, proteins, and porphyrins intomitochondria and elsewhere (1). TSPO wasfirst identified as a peripheral-type benzo-

diazepine receptor in mammals (2)—attractingattention because of popular anxiolytic benzo-diazepines such as diazepam (Valium)—and thetryptophan-rich sensory protein TspO of photo-synthetic bacteria proved to be homologous (3).The sequences of TSPO proteins from diverse or-ganisms are highly conserved in sequence (fig. S1)and also in function; notably, rat TSPO can sub-

stitute for the bacterial protein as a negativeregulator of photosynthesis gene expression inRhodobacter sphaeroides (4).Benzodiazepine-like chemicals, such as PK11195,

were found to bind to TSPO and regulate steroidbiosynthesis (5), and porphyrins were identifiedas endogenous ligands of rat TSPO, with highestaffinity for protoporphyrin IX (PpIX) (6). Manyfollowing studies have pursued TSPO interactionswith cholesterol and PpIX (7, 8). Most recently,the seeming essentiality of TSPO for steroido-genesis (9) has been challenged by the findingthat TSPO1−/− mice are viable and without de-fects in steroid hormone biosynthesis (10), andthe presumed role in PpIX transport (11) has beenamended by the discovery that bacterial TSPOscatalyze a photooxidative degradation of PpIX (12).Although the underlying biochemical mecha-

nisms for its activities are not fully understood,TSPO has elicited considerable interest for med-icine. Roles for TSPO have been imputed inapoptosis (13), inflammation (14), HIV biosynthesis(15), cancer (16), Alzheimer’s disease (17), andcardiovascular diseases (18). TSPO is a therapeutictarget (13, 18–20) and has proven useful as a pos-

itron emission tomography–imaging biomarker(14, 17, 21, 22).The translocator protein has been character-

ized in monomer, dimer, and higher oligomericstates (20). An electron microscopy structure forTSPO from R. sphaeroides (RsTSPO) revealed adimer (23), and a nuclear magnetic resonancestructure for a mouse TSPO showed a monomercomprising five transmembrane helices (24).We produced recombinant TSPOproteins from

several bacterial and vertebrate organisms andfound Bacillus cereus TSPO (BcTSPO) suitablefor biochemical and structural characterization(see supplementary materials and methods). Bysize-exclusionchromatography,detergent-extractedBcTSPO exists in at least three different oligomericstates (fig. S2A). Apo BcTSPO crystallized from themonomer-dimer fraction (fig. S2, C andD) in twodifferent lipidic cubic phase (LCP) conditions andalso from the high-oligomer fraction as a deter-gent micelle; the BcTSPO-PK11195 complex crys-tallized as a dimer in LCP. A structure obtainedby single-wavelength anomalous diffraction anal-ysis from iodinated BcTSPO (fig. S2E) was thestarting point for native structures that followed(tables S1 and S2). Refinements yielded structuresat 1.7 Å (Fig. 1, A to E, and fig. S3) and 2.0 Å res-olution for apo monomers in LCP, at 4.1 Å reso-lution for the apo dimer in detergent (Fig. 1F),and at 3.5 Å resolution for the dimeric ligandcomplex in LCP (Fig. 1G).Each BcTSPO protomer has five transmem-

brane (TM) a helices organized in clockwise order(TM1-TM2-TM5-TM4-TM3), as viewed from theC-terminal end (Fig. 1, A and B, and fig. S1) wherea short extra-membranous helix (a1,2) betweenTM1 and TM2 caps a C-terminal pocket (Fig. 1C).The electrostatic potential surface defines atransmembrane orientation (Fig. 1C) having itsN-terminal endheavily positive, likely cytoplasmicin B. cereus, and its C-terminal end largely neg-ative (Fig. 1D and fig. S4, A to D). A mapping ofsequence conservation onto themolecular surfaceshows a conservative band between TM2 andTM5, a highly conserved pocket opening betweenTM1 and TM2, and features more conserved atthe C-terminal end than on most of the exterior(Fig. 1E and fig. S4, E to H).

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1Department of Biochemistry and Molecular Biophysics,Columbia University, New York, NY 10032, USA. 2The NewYork Consortium on Membrane Protein Structure(NYCOMPS), New York Structural Biology Center, 89Convent Avenue, New York, NY 10027, USA. 3New YorkStructural Biology Center, Synchrotron Beamlines,Brookhaven National Laboratory, Upton, NY 11973, USA.4Department of Informatics, Bioinformatics andComputational Biology, Technische Universität München,Garching 85748, Germany. 5Department of Physiology andCellular Biophysics, Columbia University, New York, NY10032, USA.*Corresponding author. E-mail: wayne@xtl.cumc.columbia.edu

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Replication-transcription switch in human mitochondriaKaren Agaronyan, Yaroslav I. Morozov, Michael Anikin and Dmitry Temiakov

DOI: 10.1126/science.aaa0986 (6221), 548-551.347Science 

, this issue p. 548Sciencetranscription. These mutually exclusive mechanisms allow the processes to proceed independently as needed by the cell.primers are not formed, and the overall processivity of mtRNAP elongation complexes is enhanced, promoting genome mtRNAP terminates downstream from the promoter, forming primers to promote replication. In the presence of TEFM, thethe mitochondrial transcription elongation factor TEFM serving as a key player in the switch. In the absence of TEFM,

now show that transcription and replication are kept separate in human mitochondria, withet al.replication. Agaronyan single mitochondrial RNA polymerase (mtRNAP) transcribes the mitochondrial DNA and also generates primers for

Because mitochondrial DNA is circular, the transcription and replication machinery might be expected to collide. ASwitching transcription and replication

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