In Vitro Activities of the Multifunctional RNA SilencingPolymerase QDE-1 of Neurospora crassa*□S

Received for publication, April 28, 2010, and in revised form, July 13, 2010 Published, JBC Papers in Press, July 20, 2010, DOI 10.1074/jbc.M110.139121

Antti P. Aalto‡1, Minna M. Poranen‡, Jonathan M. Grimes§, David I. Stuart§, and Dennis H. Bamford‡2

From the ‡Institute of Biotechnology and Department of Biosciences, Biocenter 2, P.O. Box 56, University of Helsinki,FIN-00014 Helsinki, Finland and the §Division of Structural Biology, The Henry Wellcome Building for Genomic Medicine,Oxford University, Oxford OX3 7BN, United Kingdom

QDE-1 is an RNA- and DNA-dependent RNA polymerasethat has functions in the RNA silencing and DNA repair path-ways of the filamentous fungus Neurospora crassa. The crystalstructure of the dimeric enzyme has been solved, and the fold ofits catalytic core is related closely to that of eukaryotic DNA-de-pendent RNA polymerases. However, the specific activities ofthis multifunctional enzyme are still largely unknown. In thisstudy,we characterized the in vitro activities of theN-terminallytruncated QDE-1�N utilizing structure-based mutagenesis.Our results indicate that QDE-1 displays five distinct catalyticactivities, which can be dissected by mutating critical aminoacids or by altering reaction conditions. Our data also suggestthat the RNA- and DNA-dependent activities have differentmodes for the initiation of RNA synthesis, whichmay reflect themechanism that enables the polymerase to discriminate be-tween template nucleic acids. Moreover, we show that QDE-1 isa highly potent terminal nucleotidyltransferase. Our resultssuggest that QDE-1 is able to regulate its activity mode depend-ing on the template nucleic acid. This work extends our under-standing of the biochemical properties of the QDE-1 enzymeand related RNA polymerases.

Gene expression of most eukaryotic organisms is regulatedby an immense assortment of small RNAs and proteins thatassociate with them. These various components form networksknown as RNA silencing pathways, most important of whichemploy small interfering RNAs (siRNAs), microRNAs, or piwi-interacting RNAs to achieve sequence specificity (1–3). RNAsilencing associated cell-encoded RNA-dependent RNA poly-merases (RdRPs)3 are found commonly as components of theRNA silencing pathways of plants, fungi, and nematodes (1, 4).

The functions of cellular RdRPs have been largely elusive, butrecent studies have shed some light on their enigmatic charac-ter. Caenorhabditis elegans RdRPs have been shown to synthe-size secondary siRNAs that are important for amplifying theinitial silencing signal (5–7). Tetrahymena termophila RdRP(Rdr1) is known to interact with Dicer to produce endogenoussiRNAs, whereas the RdRP (Rdp1) of Schizosaccharomycespombe is critical for heterochromatic gene silencing (2, 8, 9).Arabidopsis thaliana has six genes that code for RdRPs, butonly a few of these have been studied in detail (10, 11). Most ofthe above studies imply that the main function of cellularRdRPs is in synthesizing siRNAs directly or making double-stranded RNA (dsRNA) from single-stranded RNA (ssRNA)templates to be used as Dicer substrate. For a long time, it wasthought that cellular RdRPs are absent in insects andmammals,but recently, robust RdRP activities have been detected inDro-sophila melanogaster and humans (12, 13) suggesting that cel-lular RdRPsmay have crucial functions throughout the eukary-otic domain.Neurospora crassa is a filamentous fungus that displays re-

markable genomic stability (14, 15). One of the cellular mech-anisms that affect to this stability is an RNA-silencing pathwayknown as quelling (16). Quelling is initiated by repetitivegenetic elements and is dependent on three genes: qde-1 (quell-ing defective) encoding an RdRP, qde-2 (a member of the Argo-naute family), and qde-3 (a RecQ-likeDNAhelicase) (17–19). Ithas been shown that overexpression of QDE-1 results inincreased silencing and that expression of hairpin dsRNAmol-ecules abolishes the need of QDE-1 activity, suggesting that theprimary function ofQDE-1 is to synthesize dsRNA to be used assubstrates for the two Dicers (DCL-1 and DCL-2) of Neuros-pora (15, 20–25). The Argonaute protein QDE-2 has sliceractivity and interacts with an exonuclease known as QIP (25).The expression of QDE-2 is induced by dsRNA, and its steady-state levels are regulated by the DCLs (15). The biochemicalroles of QDE-3 largely are unknown, but it has been suggestedto have roles in both DNA repair and quelling (18, 26, 27).The classical model of transgene quelling in Neurospora

begins by RNA polymerase II and QDE-3 synthesizing an aber-rant RNA molecule, which is recognized by QDE-1 and con-verted into dsRNA (24). This dsRNA is digested to double-stranded siRNAs by the DCLs. These associate with QDE-2,which nicks the passenger strand of the siRNA that is degradedsubsequently by the QIP exonuclease (25). QDE-2, now con-taining a single-stranded siRNA guide strand, finds its com-plementary mRNA (or aberrant RNA) targets, which leads to

* This work was supported by the Academy of Finland Finnish Centre ofExcellence Programme 2006-2011 (1129684, to D. H. B.) and by theUnited Kingdom Medical Research Council and SPINE2COMPLEXES(LSHGCT-2006-031220).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Table S1–S4, Figs. S1–S4, and additional references.

1 Fellow of the Helsinki Graduate School in Biotechnology and MolecularBiology.

2 To whom correspondence should be addressed: Institute of Biotechnologyand Dept. of Biosciences, Biocenter 2, P.O. Box 56, University of Helsinki,FIN-00014 Helsinki, Finland. Tel.: 358-0-9-191-59100; Fax: 358-0-9-191-59098; E-mail: dennis.bamford@helsinki.fi.

3 The abbreviations used are: RdRP, RNA-dependent RNA polymerase; DdRP,DNA-dependent RNA polymerase; ds, double-stranded; ss, single-stranded; TNTase, terminal nucleotidyltransferase; nt, nucleotide(s);qiRNA, QDE-2-interacting small RNA.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 38, pp. 29367–29374, September 17, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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the silencing of both transgenic and endogenous transcripts.Recently, this model has been challenged by the discovery thatquelling components have essential roles in the nucleus associ-ated with DNA repair (27, 28). QDE-1 was shown to co-purifywith ssDNA binding replication protein A (RPA), and DNAdamage was shown to induce QDE-2 expression. Immunopre-cipitation of QDE-2 from DNA damaged Neurospora culturesrevealed a novel type of small RNAs known as QDE-2-interact-ing small RNAs that are mostly derived from the ribosomalDNA (rDNA) locus (27). qiRNA production is dependent onQDE-1, QDE-3, and the DCLs but not on QDE-2. Notably,QDE-2-interacting small RNAs are derived from aberrantRNAs that are synthesized by QDE-1 and not by any of thecanonical RNA polymerases. QDE-1 was shown to have arobust DNA-dependent RNApolymerase (DdRP) activity, gen-erating a DNA/RNA hybrid from an ssDNA template (27).Much insight into the structure and function of cellular

RdRPs has come from the studies of a recombinant QDE-1 andits catalytically active C-terminal portion QDE-1�N (residues377–1402 of the wild-type) (29–31). The recombinant poly-merase is able to initiate RNA synthesis without a primer andconvert heterologous ssRNAs into double-stranded molecules.In addition to making full-length dsRNA copies of ssRNA tem-plates, QDE-1 was observed to synthesize small 9–21-nt RNAsscattered along template RNAs (29). The crystal structure ofQDE-1�N showed that the molecule is a dimer and that thecatalytic core has a fold that is related to those in eukaryoticDdRPs (31).In this study, we demonstrate that QDE-1�N displays five

distinct in vitro activities. We use structure-based mutagenesisto show that the activities can be dissected by mutating criticalamino acid residues and suggest that RdRP and DdRP activitieshave different initiation mechanisms and pH optima. The bio-chemical data presented in this study imply a recognitionmechanism that discerns a DNA template from an RNA tem-plate. These results have broader ramifications in eukaryoticRNA- and DNA-dependent RNA polymerases associated withRNA silencing pathways.

EXPERIMENTAL PROCEDURES

Site-directed Mutagenesis—The expression vectors forQDE-1�N point mutants were generated by site-directedmutagenesis using PCR. The mutagenic primers are listed insupplemental Table S1. Plasmid pEM69 (30) encoding for aHis-taggedQDE-1�N (missing amino acids 1–376) was used asa template in 50-�l PCR reactions each containing sense andantisense primers, and 2.5 units of PfuTurbo DNA polymerase(Stratagene). After completion, the reactions were treated with10 units of DpnI (Fermentas) and transformed into CaCl2 com-petent Escherichia coliXL1-Blue cells (Stratagene). The correctconstructs were verified by restriction enzyme analysis andsequencing.Yeast Expression andProtein Purification—The recombinant

proteins were expressed and purified as described previouslyfor QDE-1�N (pEM69; (30)). Briefly, the plasmids were intro-duced into Saccharomyces cerevisiae strain INVSc1 (Invitro-gen), the recombinant proteins were expressed at �28 °C for22 h and purified to near homogeneity. The yeast cells were first

harvested and disrupted by a French press. The cell lysates werethen cleared by centrifugation, and the supernatants wereloaded onto nickel-nitrilotriacetic acid affinity columns (Qia-gen), washed with 5 mM and 25 mM imidazole-containing buff-ers, and eluted with 200 mM imidazole. Subsequently, the pro-teins were purified by HiTrapTM heparin HP and Q HPcolumns (GE Healthcare) and eluted by increasing NaCl gradi-ents. The purified proteins were stored in 50 mM Tris-HCl (pH8.0), 0.1 mM EDTA, 0.13% Triton X-100, 100 mM NaCl, and62.5% glycerol at �20 °C. The oligomeric status of the recom-binant polymerases was analyzed by size-exclusion chromatog-raphy using a Superdex 200 16/60 gel filtration column (GEHealthcare) with appropriate control proteins (Sigma).Template RNAs and DNAs—Plasmid pLM659 (32) contains

a cDNA copy of the S segment of bacteriophage �6 under a T7promoter. For the production of ssRNA, pLM659 was linear-ized by SmaI digestion, purified using a PCR purification kit(Qiagen), and used as a template for run-off transcription by T7RNA polymerase. The template DNAwas degraded with DNa-seI (Promega) and the ssRNApurified by chloroformextractionand LiCl precipitation. To generate a ssDNA molecule of thesame length and sequence, SmaI-digested pLM659 was used asa template in PCR reactions containing primers AO49 andAO50 (see supplemental Table S4) and Phusion� DNA poly-merase (Finnzymes). AO50 contains a 5�-biotin. The biotinylatedPCR product was immobilized onto Dynabeads� MyOneTM

streptavidin C1 magnetic beads (Invitrogen) according to themanufacturer’s instructions. The immobilized PCR productwas dissolved by treating the DNA briefly with fresh 0.1 M

NaOH. The ssDNA was precipitated with sodium acetateand ethanol and gel-purified through agarose gel electro-phoresis. Prior to 5�-labeling, the ssRNA was treated withalkaline phosphatase (Finnzymes). Both ssRNA and ssDNAwere 5�-labeled with [�-32P]ATP (NEN Radiochemicals,PerkinElmer Life Sciences) and T4 polynucleotide kinase (Fer-mentas). M13mp18 ssDNA was purchased from New EnglandBiolabs. The oligonuleotides (AO49–52) were purchased frombiomers.net or Eurofins MWGOperon.Polymerase Activity Assays—Polymerase reactions were per-

formed essentially as described (29, 33). The standard QDE-1reaction mixture contained 50 mM HEPES-KOH (pH 7.8), 20mM ammonium acetate, 1 mM MgCl2, 1 mM MnCl2, 6% (w/v)polyethylene glycol 4000, 0.1mMEDTA, 0.1%TritonX-100, 0.2mM of each NTP, 1 unit/�l RNasin� ribonuclease inhibitor(Promega), and 0.01–0.02 �g/�l QDE-1�N. In some of the pHexperiments, HEPES-KOH (pH 7.2–7.8) was replaced by Bis-Tris (pH 6.0–6.9) or Tris-HCl (pH 8.0–8.9). The ladder reac-tions were programmedwith 5mMMgCl2. Reactions were sup-plemented with 0.1 mCi/ml of [�-32P]UTP (GE Healthcare orNEN Radiochemicals, PerkinElmer Life Sciences) or otherradioactive NTPs where indicated. The reactions were incu-bated at�30 °C for 1 h and quenched with U2 (8 M urea, 10mM

EDTA, 0.2% SDS, 6% (v/v) glycerol, 0.05% bromphenol blue,and 0.05% xylene cyanol) loading buffer. Some reaction prod-ucts were extracted with phenol:chloroform:isoamyl alcohol(25:24:1) and chloroform:isoamyl alcohol (24:1), precipitatedwith NH4OAc and ethanol, and dissolved in milli-Q water.

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Some samples were treated with RNase T1 (Fermentas) for 15min at �37 °C.The samples were subjected to standard Tris-Borate-EDTA

or Tris-Acetate-EDTA agarose gel electrophoresis or denatur-ing, formaldehyde-containing agarose gel electrophoresis (34).The gels were visualized by ethidium bromide staining anddried, and radioactivity was detected by phosphorimaging (FujiFLA-5000) and analyzed by densitometry with AIDA software(Raytest Isotopenme�gerate). Some samples were subjected todenaturing PAGE by urea-containing 20% sequencing gels.These were either desalted by Zeba Spin Desalting Columns(Thermo Scientific) or purified by phenol extraction and etha-nol precipitation. Prior to loading, the samplesweremixedwithGel Loading Buffer II (Ambion) and heated to�95 °C for 5min.

RESULTS

QDE-1 Displays Five Distinct Activities—To elucidate thedifferent catalytic activities ofQDE-1�N, standard polymeriza-tion reactions were carried out with ssRNA or ssDNA tem-plates in different conditions (Fig. 1A). Mixing QDE-1�N withssRNA, all four NTPs and [�-32P]UTP resulted in dsRNA syn-thesis as well as decreasedmobility (shifting) and labeling of thessRNA template (lane 2). In addition, labeled RNA productswere detected that varied in size from tens of nucleotides toseveral hundreds. Some of these products did not enter theagarose gel and remained in thewells. No labeled productsweredetected when the catalytically inactive QDE-1�NDA was usedin the reaction mix (lane 3) (29). When the reactions were car-ried out with only UTP and trace amounts of [�-32P]UTP, thetemplate ssRNAwas efficiently labeled without dsRNA synthe-sis (lane 4), indicating that QDE-1�N is a potent terminalnucleotidyltransferase (TNTase). QDE-1�N displays also astrong DNA-dependent RNA polymerase activity (lane 6) (27).None of the template ssDNA migrates as template-sized but isvery efficiently converted to the DNA/RNA hybrid form (lane6). Again, substituting the �N polymerase with QDE-1�N DAabolishes this activity (lane 7). The TNTase activity also is veryprominentwith an ssDNA template (lane 8). Using onlyUTP asthe substrate, the ssDNA template migrates at its normal posi-tion (upper panel) but is extensively labeled (lower panel).The above experiments show that QDE-1�N displays five

distinct activities (Fig. 1, A and B): (i) RNA-dependent RNApolymerase activity (Fig. 1A, lane 2) (29), (ii) DNA-dependentRNA polymerase activity (Fig. 1A, lane 6) (27), (iii) ssRNA tem-plate shift and labeling activity (Fig. 1A, lane 2), (iv) TNTaseactivity (Fig. 1A, lanes 4 and 8), and (v) ladder activity (Fig. 1A,lanes 2, 6, and 9) (supplemental Fig. S1). Activities (i) and (ii)have been described previously (27, 29). Activity (iii) has beensuggested previously to result from the synthesis of 9–21-ntsmall RNAs that are scattered across the ssRNA template, aswell as to be the main in vitro reaction product of QDE-1�N(29). However, in this work, the intensity of the ssRNA labelingwas not significantly more extensive than the labeling of thedsRNA product (Fig. 1A, lane 2), suggesting that the “smallRNAs” are not the main reaction product. The identity of thisactivity is further discussed below. TNTase activity (iv) (seebelow) has been detected previously in both viral and eukary-otic RdRPs, where nucleotides are added to the 3�-ends of the

templates in a template-independent fashion (11, 35). Activity(v) (Fig. 1A and supplemental Fig. S1) was assigned as a ladderactivity because it generates RNA products of all sizes. Whenthe reaction products of a reaction without a template wereanalyzed on a denaturing sequencing gel, they migrated at one-nucleotide increments (starting from �8 nts) forming a “lad-der” (supplemental Fig. S1A). This activity is template-inde-pendent because omitting the template does not affect theformation of the ladder (Fig. 1A, lane 9). However, the sensitiv-ity of the ladder activity to varying reaction conditions suggeststhat it is an in vitro side reaction occurring at high enzyme andsubstrate conditions (supplemental Fig. S1). The in vitro activ-ities of QDE-1�N are summarized schematically in Fig. 1B.QDE-1 Uses Different Initiation Mechanisms on ssDNA and

ssRNA Templates—As QDE-1 displays both RNA- and DNA-dependent RNA polymerase activities, we further studied thenature of these reactions. We programmed polymerization

FIGURE 1. QDE-1 displays five distinct activities. A, QDE-1 reactions wereprogrammed with the same amounts of ssRNA or ssDNA, NTPs or UTP, andQDE-1�N or QDE-1�NDA as indicated. All reactions contained trace amountsof [�-32P]UTP. The control nucleic acids were labeled in the 5�-end with[�-32P]ATP and polynucleotide kinase. Reaction mixtures were incubated for1 h at �30 °C, quenched with U2 loading dye, and analyzed by native agarosegel electrophoresis. Upper panel, ethidium bromide-stained gel; lower panel,autoradiogram of the same gel. Positions of templates (ss) and products (ds)are indicated. The band migrating in between ss and ds on lanes 5, 7, and 8 isa conformer of the ssDNA template. B, a schematic presentation of differentQDE-1 in vitro activities. See text for details.

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reactions with ssRNA or ssDNA templates of the same lengthand sequence, purified the reaction products, and analyzedthem by denaturing formaldehyde-containing agarose gel elec-trophoresis. As controls we labeled the templates at the 5�-endwith [�-32P]ATP and polynucleotide kinase (Fig. 2A). As hasbeen shown previously (29), most of the product that accumu-lated using an ssRNA template migrated more slowly than thetemplate, indicating that the strands of the dsRNAmolecule arecovalently linked together (“back-priming”). In contrast, theproducts of the reaction using an ssDNA template migrated astemplate-sized or smaller, indicating that the mode of RNAsynthesis initiation differs between these two templates. Thisresult is further supported by an experiment where RdRP andDdRP reactions were carried out with an initiating nucleotidethat was 32P-labeled at the �-phosphate (Fig. 2B). In this exper-imental setup, the product RNA can be labeled only if the�-phosphate remains within the first nucleotide of the newstrand. Conversely, if RNA synthesis is initiated by back-prim-ing, the �-phosphate is removed from the product RNA. Asexpected, radioactivity was detected only in the double-stranded products of the ssDNA template (DNA/RNAhybrids)and the template-sized products of the ssRNA template (result-ing from abortive initiation, see below). dsRNAwas not labeled.In addition, we performed QDE-1�N activity assays with bothssRNA and ssDNA templates in the same reaction mixturesimultaneously (supplemental Fig. S2). The RdRP or DdRPactivities did not inhibit each other, but both templates wereprocessed into products. The ssDNA template in this experi-ment was circular, indicating that QDE-1�N is able to initiateRNA synthesis without the need for a free 3�-end (supple-mental Fig. S2). All in all, these data suggest that QDE-1 is ableto discriminate between ssRNA and ssDNA templates.Mutant Polymerases Display Altered Activities—The crystal

structure of QDE-1�N has been solved previously (31). Usingthe structural information, we designed eight point mutations

that were predicted to functionally disrupt QDE-1�N (Fig. 3Aand supplemental Fig. S3 andTables S1 and S2). The constructswere transformed into S. cerevisiae, and the recombinant pro-teins were expressed. All of the mutant enzymes were solubleand purified to near homogeneity and behaved like the wild-type during purification (data not shown). Initial screeningof the RdRP and DdRP activities of the point mutants revealedthat they possessed characteristics that differed from the wild-type polymerase (supplemental Fig. S4A). As expected (as thesewere assumed to be catalytically essential aspartic acids), QDE-1�NDA (D1011A) (29) and D1007A were catalytically com-pletely inactive. Of the active point mutants, five (R738A,R944E,K1119W,M1357D, andM1357C)were chosen formoreextensive studies due to their catalytic properties. Arg738 lieswithin a channel that is predicted to accommodate the reactionproduct of QDE-1�N and direct it away from the active site(Fig. 3A). The R944E mutation is predicted to partly block thecommunication tunnel that links the two active sites in a QDE-1�N dimer (Fig. 3A). The K1119W mutation was designed toocclude a pore in QDE-1�N that apparently allows substratenucleotides to enter the active site. The M1357D and M1357Cmutations are predicted to respectively weaken and locktogether the dimeric interface of the QDE-1�N head domains(Fig. 3A). However, the interface of the entire QDE-1 dimer isso extensive that mutating Met1357 should not affect the oligo-merization state of the enzyme. To confirm this, we performedanalytical gel filtration chromatography with QDE-1�NWT indifferent conditions, some of the point mutant enzymes, andcontrol proteins of various sizes (Table 1). Dimeric QDE-1�Nis predicted to be �230 kDa in size, whereas a monomer wouldhave the predicted size of �120 kDa (31). Our results establishthat all QDE-1�N enzymes are dimeric, regardless of the sur-rounding pH (Table 1). This has been deduced previously fromthe crystal structure, as each of the subunits has �2000 Å2 ofcontact area with the neighboring subunit (31).All of the five point mutants under closer scrutiny were

catalytically active on both ssRNA and ssDNA templates(Fig. 3B). The catalytic activity of R738A is reduced toapproximately half of that of the wild-type regardless of thetemplate (Fig. 3C), in accordance with a nonspecific chargesteering role for this residue. In addition, R738A is incapableof shifting and labeling the ssRNA template (Fig. 3B andsupplemental Fig. S4A). The DdRP activity of R944E is closeto that of the native polymerase. However, its RdRP activityis only �20% of the wild-type (Fig. 3C). It shifts the ssRNAtemplate and labels it efficiently (Fig. 3B and supplemen-tal Fig. S4A). These results are consistent with a role for thetunnel bridging the active sites of the dimer to initiate RNA-templated polymerization (see below). The RdRP and DdRPactivities of K1119W are close to those of the wild-type, theformer activity even being slightly higher (Fig. 3C). However,this mutant shows dramatically reduced activities (iii) and(iv) (shifting of the template ssRNA and TNTase activity) (Fig.3B and supplemental Fig. S4, A and B), which may reflect aslight weakening of nonspecific charge stabilization of a prod-uct complex by this residue close to the active site. BothM1357D and M1357C mutants display catalytic activities thatare very similar to wild-type (Fig. 3, B and C). The ssRNA tem-

FIGURE 2. QDE-1 has different initiation mechanisms for DNA and RNAtemplates. A, QDE-1�N reactions were programmed as described in Fig. 1and incubated for 1 h at �30 °C. The reactions were purified by phenol extrac-tion and ethanol precipitation, and the samples were subjected to denatur-ing agarose gel electrophoresis. On control (CTL) lanes are the templatenucleic acids labeled in the 5� end. Shown is the autoradiogram of the gel.B, QDE-1�N reactions were programmed with ssRNA or ssDNA with the samelength and sequence. Both templates end with CC-3�. All four NTPs and traceamounts of [�-32P]GTP were added in the reactions. The products were ana-lyzed by native agarose gel electrophoresis, and shown are EtBr-stained gel(upper panel) and autoradiogram of the same gel (lower panel).

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plate shift and labeling in M1357Dseems to be somewhat more exten-sive, and the amount of dsRNAproduct seems smaller than in thewild-type. This result suggests thatmutating a single amino acid at thedimerization interface of the headdomain has negligible effects onQDE-1 in vitro activity. In general,DdRP activity is less sensitive tomutations than RdRP activity, fur-ther suggesting that QDE-1 is pri-marily a DNA-dependent RNApolymerase (see below). The activi-ties of the different point mutantsare summarized in supplemen-tal Table S3.pH Has a Differential Effect on

RdRP and DdRP Activities of QDE-1�N—Point mutations to QDE-1structure introduce internal varia-tions to the protein. To study exter-nal factors on the catalytic proper-ties of the enzyme, we assayed thepH dependence of QDE-1�N onRdRP and DdRP activity by using alinear ssRNA or a circular ssDNA astemplates in reactions with QDE-1�N WT (Fig. 4A). To our surprise,we observed that the RdRP activitywas high at a low pH (optimum atapproximately pH 6.3) and de-creased as the pH increased. In con-trast, the DdRP activity was low at alow pH and increased with theincreasing pH (optimum at approx-imately pH 7.4). Similar results wereobtained when the QDE-1�N WTreactions were programmed withlinear ssRNA or ssDNA of the samelength and sequence at pH 6.3, 7.4,and 8.3 (Fig. 4B). The DdRP activitywas, however, always higher than

the RdRP activity. We also performed reactions with all theQDE-1�N point mutants varying the pH (Fig. 4C and data notshown). Interestingly, the K1119W mutant was catalyticallyinactive at pH 6.3 with both ssRNA and ssDNA templates,regaining its activity as the pH increased (Fig. 4C). These dataindicate that pH might be one of the factors regulating RdRPand DdRP activities.Mutations Affecting TNTase Activity Suggest a Mechanism

for Template Recognition—Terminal nucleotidyltransferaseactivity has been described in both viral and cellular RdRPs (11,35). QDE-1�N WT displays a strong TNTase activity (iv) onlinear ssRNA and ssDNA templates (Fig. 1A). To ensure thatthe detected activity truly occurs at the 3�-end of the template,we designed a 30-nt-long RNA oligonucleotide that has a singleG residue at the 10th position from the 5�-end (Fig. 5A). When

FIGURE 3. QDE-1�N point mutants display characteristics that differ from the wild-type enzyme.A, schematic presentation of the QDE-1�N dimer and the mutations introduced by site-directed mutagen-esis. Subunit A of the dimer is colored according to domains: blue, slab; purple, catalytic; pink, neck; andorange, head. Subunit B is colored gray (for domain definition, see Ref. 31), and the approximate positionsof the NTP pore, the bridging tunnel between active sites, and product channel in subunit A are high-lighted by arrows. The left panel is a view approximately orthogonal to that of the main image and showsa close-up of the active site of subunit A with the mutated residues drawn as sticks, and the Mg2� ion isshown as a cyan sphere. The right panel shows a view of the mutations (by �90 o about the vertical axis)introduced into the bridging tunnel between the active sites of the molecule. This figure was producedusing PyMOL. B, standard QDE-1 reactions were programmed with ssRNA (left panel) or ssDNA (rightpanel), NTPs, [�-32P]UTP, and equal amounts of QDE-1�N enzymes as indicated. The reaction productswere analyzed by native agarose gel electrophoresis. Upper panel, ethidium bromide-stained gel; lowerpanel, autoradiogram of the same gel. Positions of templates (ss) and products (ds) are indicated. Oncontrol (CTL) lanes are the template nucleic acids labeled in the 5� end. C, equal amounts of ssRNA orssDNA templates with the same length and sequence were used as templates in QDE-1�N reactions.Incorporated radioactivity in the products was quantified by phosphorimaging. The activity of the wild-type enzyme was set as 100%. The experiment was repeated independently three times. Error bars indi-cate the S.E.

TABLE 1Analytical gel filtrations of QDE-1�N mutant polymerasesElution was performed in 50 mM HEPES-KOH pH 7.4, 150 mM NaCl. QDE-1�NWT and alcohol dehydrogenase were also analyzed in 25 mM Bis-Tris (pH 6.3), 150mM NaCl, and 50 mM Tris-HCl (pH 8.9), 150 mM NaCl.

Protein Peak elution time

minApoferritin (Sigma),443 kDa

113.47

QDE-1�NWT 129.52 (pH 6.3), 127.76 (pH 7.4), 128.03 (pH 8.9)QDE-1�N D1007A 126.75QDE-1�N P964A 127.92QDE-1�N R738A 127.23QDE-1�NM1357D 127.23QDE-1�N K1119W 127.23QDE-1�N R944E 127.12Alcohol dehydrogenase(Sigma), 150 kDa

137.42 (pH 6.3), 137.31 (pH 7.4), 135.82 (pH 8.9)

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this oligonucleotide was labeled at the 5�-end with [�-32P]ATPand polynucleotide kinase and subsequently digested withRNase T1 (that specifically cleaves ssRNA at 3� of G residues),the labeled product was 10-nt-long (Fig. 5A, lanes 1 and 2).When an unlabeled oligonucleotide was incubated with QDE-1�N WT and [�-32P]UTP, the reaction product migrated at aposition corresponding to�31 nt. As this product was digestedwithRNaseT1, the position of the label corresponded to�21nt(Fig. 5A, lanes 3 and 4). These results indicate that QDE-1transfers approximately one nucleotide to the 3�-end of thetemplate. In addition to UTP, the other NTPs (ATP, GTP, andCTP) were accepted as substrates as well (data not shown).To further investigate the TNTase activity, we used all eight

point mutants in a TNTase assay with both ssRNA and ssDNAtemplates (supplemental Fig. S4B). QDE-1�NDA and D1007Awere completely inactive, suggesting that the TNTase activityresides in the same catalytic site as the other activities. Thepoint mutants labeled the templates with different efficiencies,with R738A and K1119W showing very little activity. To oursurprise, we noticed that the P964Amutantwas able to label thessRNA but not the ssDNA.WhenQDE-1�N structure (31) wassuperimposed with yeast RNA polymerase II elongation com-plex (based on the conserved double-psi �-barrels) the incom-ing DNA template could be modeled very close to Pro964 (36and data not shown). Mutation P964A introduces changes tothe course of the polypeptide chain in its proximity but isfar enough from the active site (11 Å) to produce only a subtleeffect. When QDE-1�N WT and P964A polymerases werecombined with both template types and NTPs or UTP, theP964A mutant differed from the �N WT only in being un-able to add a terminal UTP to the 3�-end of the ssDNA (Fig. 5B).We also performed the TNTase assays with 30-nt-long ssRNAor ssDNA oligonucleotides and analyzed the reaction productson a denaturing polyacrylamide gel (Fig. 5C). The �NWT andP964A enzymes both added �1 nucleotide on the 3�-end of thessRNA. The �N WT also was capable of adding �1 nt on the3�-end of the ssDNA template, albeit less efficiently, whereasthe P964A mutant was inactive when the ssDNA template wasapplied. These results indicate thatQDE-1 is able to distinguishbetween ssRNA and ssDNA. Although the ssRNA and ssDNAoligonucleotides have the same sequence, the ssRNA migratedmore slowly on the denaturing gel than the ssDNA due to itshighermolecularweight (9189 g/mol versus 8821 g/mol). As themolecular weight of uridyl monophosphate is �324 g/mol, thisresult shows that in these conditions QDE-1 adds only onenucleotide to the 3�-end of the template. However, in higherNTP concentrations, the number of added nucleotides mayincrease (data not shown).

DISCUSSION

In this study, we have shown that QDE-1 displays fivedistinct activities on RNA or DNA templates (Fig. 1) and thatthese activities can be dissected by altering the reaction con-ditions (Fig. 4) or utilizing mutant polymerases (Fig. 3).Interestingly, both DdRP and TNTase activities also havebeen described for RDR6 of Arabidopsis (11), suggesting thatsuch biochemical activities may be conserved evolutionallyamong cellular RdRPs. Our results indicate that QDE-1 is most

FIGURE 4. pH has an effect on RdRP and DdRP activities. A, a linear ssRNAand a circular ssDNA were used as templates in QDE-1�N WT reactionscontaining 25 mM Bis-Tris (pH 6.0 to 6.9), 50 mM HEPES-KOH (pH 7.2 to 7.8)or 50 mM Tris-HCl (pH 8.0 to 8.9) in addition to all the standard compo-nents. The maximum (max.) activity of each reaction set (RdRP or DdRP)was set as 100%. The experiment was repeated independently threetimes. Error bars indicate S.E. B, equal amounts of ssRNA or ssDNA tem-plates with the same length and sequence were used as templates inQDE-1�N WT reactions in the indicated pH values. The maximum activityof each reaction set (RdRP or DdRP) was set as 100%. The experiment wasrepeated independently three times. Error bars indicate S.E. C, equalamounts of QDE-1�N WT or K1119W were used in reactions similar asabove. Shown are the autoradiograms of native agarose gels.

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effective in vitro as aDdRP and that the initiationmechanismofRNA synthesis is different with ssRNA and ssDNA templates(Fig. 2 and supplemental Fig. S2).

It was suggested previously that the template shift (iii) seen inthe RdRP reaction of QDE-1 results from the synthesis of smallRNAs of 9–21 nt that are scattered across the template ssRNA(29). Subsequent studies have, however, shown that themain invivo functions of QDE-1 are aberrant RNA (on a DNA tem-plate) and dsRNA (on an RNA template) synthesis, and the“small RNAs” may not be relevant biologically (15, 20, 27).Indeed, when dsRNA is produced from hairpin constructs inNeurospora, the requirement for QDE-1 is abolished, but RNAsilencing is not compromised (15, 20). Our results suggest thatthe template shift is not required for efficient dsRNA synthesis(see, for example, R738A, R944E, and K1119W in Fig. 3B), andit is not detectedwhenusing an ssDNA template. Thus, it seemsplausible that this shift is not an essential step in the reactionpathway leading to dsRNA production but rather a side reac-tion resulting from abortive RNA polymerization initiation.Based on the biochemical and genetic evidence obtained

here, we suggest a model for QDE-1 activity in vitro. It is plau-sible that the dimeric nature of the polymerase is crucial forits activity; QDE-1�N is a functional dimer, and the twosubunits, tightly associated with each other, have slightly

different conformations (31). Therealso is a distinct communicationtunnel connecting the catalytic sitesof the two subunits (31; see also Fig.3A). Mutation R994E partly blocksthis tunnel resulting in significantreduction in the RdRP activity (i)but with no effect on the DdRPactivity (ii) (Fig. 3B). We thereforepropose that the DdRP activity ofQDE-1 is a “monomeric” feature ofthe enzyme, whereas for RdRPactivity, the ssRNA template wouldhave to be guided through the com-munication tunnel to the active siteof the other subunit for dsRNA syn-thesis, making the RdRP activity a“dimeric” property of the enzyme.Indeed, in the structurally similaryeast DdRP elongation complex,there is no 2-fold symmetry (36) sup-porting the idea that theDNA-depen-dent reaction of QDE-1 would occurindependently at the two active sites.The different pHoptima (Fig. 4), thedifferent initiationmodes (Fig. 2), aswell as the observation that the twoactivities may occur simultaneously(supplemental Fig. S2) further sup-port the idea that the RdRP andDdRP activities are uncoupled.DdRP activity is likely the pri-

mary activity of QDE-1 as it is con-stantly considerably higher than

the RdRP activity (Figs. 1 and 3B). Apparently, the RNA mole-cules also are directed via the “DdRP” site through the commu-nication tunnel to the “RdRP” site of the dimer.However, not allssRNA molecules would reach the RdRP site at the other sub-unit under the in vitro conditions applied. They would belabeled erroneously by the DdRP active site, which would resultin an abortive product that then appears as activity (iii) (asexemplified by R944E in Fig. 3B and labeling of ssRNA in Fig.2B). Such small RNAs that were predicted previously to causethe shifting of the ssRNA template (29) have not been observedin vivo (15, 20, 27).It is possible that the TNTase activity (iv) would target an

ssRNA template on the other subunit for dsRNA synthesis.This is supported by the notion that the TNTase activity in theP964Amutant labels only ssRNA but not ssDNA. Themutatedproline lies close to the active site and might convey the tem-plates in proper directions. The incoming DNA templateapparently passes very close to residue P964 indicating that itmight be expected to impact the discrimination of RNA andDNA templates. This is precisely what was observed biochem-ically (Fig. 5, B and C).As pH has a clear differential effect on the RdRP and DdRP

activities of QDE-1, it may be that this would reflect pH differ-ences in various cellular compartments (e.g. nucleus and cyto-

FIGURE 5. The terminal nucleotidyl transferase activity of QDE-1. A, sequence and RNase T1 cleavageproducts of the 30-nt-long ssRNA (upper panel). Control and QDE-1�N reactions were purified by phenolextraction and ethanol precipitation and analyzed on a denaturing 20% urea-PAGE (lower panel). The bandmigrating at �20 nt on lane 1 is a conformer of the ssRNA. B, standard QDE-1 reactions were programmed withssRNA or ssDNA, NTPs or UTPs, [�-32P]UTP and equal amounts of QDE-1�N enzymes as indicated. The reactionproducts were analyzed by native agarose gel electrophoresis, and shown is the autoradiogram of the gel.Positions of single- and double-stranded nucleic acids are indicated. C, TNTase reactions with QDE-1�N WT orP964A were programmed with unlabeled 30-nt-long ssRNA (AO51) or ssDNA (AO52) and [�-32P]UTP. Thereaction products were purified by phenol extraction and ethanol precipitation and analyzed on a denaturing20% urea-PAGE. Control (CTL) lanes contain the oligonucleotides labeled to the 5�-ends.

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plasm). However, because the QDE-1 dimer is known to asso-ciate with several proteins and other cellular factors (15, 27) it isobvious that there are many regulatory mechanisms that con-trol the activity of this multifunctional polymerase.This work lays the biochemical foundations for the proper-

ties of QDE-1. Consequently, it is possible to apply this knowl-edge in future in vivo studies, allowing us to probe the effect ofamended polymerases in quelling and other activities.

Acknowledgments—We thank Riitta Tarkiainen, Satu Hyvarinen,Xiaoyu Sun, and Sampo Vehma for excellent technical assistance.

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1

SUPPLEMENTAL DATA

Figure S1. The ladder reaction is sensitive to varying reaction conditions.

(A) A standard QDE-1 reaction was programmed without a template and incubated at +30ºC for two

hours. The reaction was desalted, denatured by heat and analyzed on a 20 % urea-containing

polyacrylamide gel. Positions of 1 nt and 30 nts are indicated on the left.

(B) and (C) Equal amounts of ssRNA or ssDNA templates with the same length and sequence were

used as templates in QDE-1 N WT reactions at the indicated NaCl concentrations (B) or

temperatures (C). The templates were omitted in ladder reactions. The maximum activity of each

reaction set (RdRP, DdRP or ladder) was set as 100 %. The experiments were repeated independently

two times. Error bars indicate the standard errors of the mean.

(D) Standard QDE-1 reactions were programmed without a template. NTPs and trace amounts of

corresponding -32P-NTPs were added as indicated. The reaction products were analyzed by native

agarose gel electrophoresis, and shown is the autoradiogram of the gel. The ladder occurs only with

combinations of ATP and UTP.

Figure S2. RdRP and DdRP reactions can occur simultaneously.

Standard QDE-1 reactions were conducted with a linear ssRNA template, a circular ssDNA template

and QDE-1 N WT. In each reaction set the concentrations of two variables were kept equimolar,

while the third was added incrementally as indicated. Titration of (A) QDE-1 N WT, (B) ssDNA and

(C) ssRNA. The reactions were incubated at +30ºC for one hour and the reaction products were

analyzed by native agarose gel electrophoresis. Upper panels: autoradiograms of the gels. The faint

band between the DNA/RNA and dsRNA bands is a conformer of the DNA/RNA product. Lower

panels: the incorporated radioactivity was quantified for each reaction product and shown are plots of

increasing relative concentration of (A) QDE-1 N WT, (B) ssDNA and (C) ssRNA as a function of

2

relative QDE-1 activity (% of maximal value, filled circles: dsRNA, open circles: DNA/RNA hybrid).

Shown are the means of two independent experiments. Error bars indicate the standard deviations.

Figure S3. Mutation sites within QDE-1 sequence.

The figure shows the amino acid sequence of wild-type QDE-1 (accession number EAA29811). The

sequence of QDE-1 N is in bold, and the missing N-terminus is overlined in gray. The mutated

amino acids are depicted in red and the mutations are indicated above the residues. Three subdomains

(DPBB1, DPBB2 and FLAP) of the catalytic domain are outlined by cyan boxes. DPBB is an

abbreviation from double-psi -barrel.

Figure S4. Overview of the activities of the point mutant enzymes.

(A) A linear ssRNA and a circular ssDNA were used as templates in standard QDE-1 N reactions.

As a control, the ssRNA was labeled in the 5’ end. The samples were analyzed by native agarose gel

electrophoresis. Upper panel: ethidium bromide stained gel, lower panel: autoradiogram of the same

gel.

(B) Terminal nucleotidyl transferase (TNTase) activity of QDE-1 N enzymes was analyzed by

mixing them with cold UTP, -32P-UTP and linear ssRNA or ssDNA of the same length and

sequence. The samples were analyzed by native agarose gel electrophoresis and shown are

autoradiograms of the gels.

3

Table S1. Plasmids and mutagenesis primer sequences.

Plasmid

name

Mutation Sense primer Antisense primer

pAA8 D1007A CTTGCTAAGAAGCTTTCTGGTGGAGCCT

ACGACGGCGATATGG

CCATATCGCCGTCGTAGGCTCCACCAGAAAGCT

TCTTAGCAAG

pAA9 P964A GTCCTCGTGGCGCGATCGGCAGCCCATT

TCCCTAGTGATATC

GATATCACTAGGGAAATGGGCTGCCGATCGCG

CCACGAGGAC

pAA10 R1091A GCATGTGCACTAACTACAAAGAAGCGC

TCTGTTACATCAACAATAGTG

CACTATTGTTGATGTAACAGAGCGCTTCTTTGT

AGTTAGTGCACATGC

pAA11 R738A GATGTGCCCTCTGCAGTGCAAGGGGCG

TTTGGTTCGGCCAAG

CTTGGCCGAACCAAACGCCCCTTGCACTGCAGA

GGGCACATC

pAA12 M1357D GTTCATGTATGCGGGCTTGGACCCGGAT

AAGAAGTTTACGAAG

CTTCGTAAACTTCTTATCCGGGTCCAAGCCCGC

ATACATGAAC

pAA13 M1357C GTTCATGTATGCGGGCTTGTGCCCGGAT

AAGAAGTTTACGAAG

CTTCGTAAACTTCTTATCCGGGCACAAGCCCGC

ATACATGAAC

pAA14 K1119W GAAACCTCGTCGATCAGAGCTGGCAAG

GTATTGTCTTTAACG

CGTTAAAGACAATACCTTGCCAGCTCTGATCGA

CGAGGTTTC

pAA15 R944E CATGTCGGATTCTCATCAAAGTTCGAGG

ACGAGGAGGAGTCTTTTAC

GTAAAAGACTCCTCCTCGTCCTCGAACTTTGAT

GAGAATCCGACATG

Table S2. Predicted mutations of the QDE-1 enzymes.

Plasmid name Mutation Mutation site

pEM69 None None

pEM56 D1011A Active site

pAA8 D1007A Active site

pAA9 P964A Active site

pAA10 R1091A Incoming RNA

pAA11 R738A Channel RNA away

pAA12 M1357D Dimer helical head domain interface

pAA13 M1357C Dimer helical head domain interface

pAA14 K1119W Block nucleotide pore entrance

pAA15 R944E Communication tunnel

4

Table S3. Activities of the QDE-1 enzymes.

QDE-1 enzyme RdRP (i) DdRP (ii) RNA shift (iii) TNTase (iv) Ladder (v)

N WT ++ +++ ++ +++ +++

R738A + + - + -

R944E + +++ +++ ++ -

K1119W +++ (pH*) +++ (pH*) - + -

M1357D + +++ +++ +++ +++

M1357C ++ +++ ++ +++ +++

P964A ++ ++ + ssRNA ++

ssDNA - -

* K1119W is inactive at a low pH and regains its activity as pH increases.

Table S4. Primer sequences.

Primer name Primer sequence

AO#49 5´- ACTCTTATATAAGTGCCCTTAGC -3´

AO#50 5 - BIO-GGTCCTATTGGACGCTC -3´

AO#51 (RNA) 5´- CCCUACCCCGCCCUAUUUCCCCCUUUCCCC -3´

AO#52 (DNA) 5´- CCCTACCCCGCCCTATTTCCCCCTTTCCCC -3´

B

C

ATPGTPCTPUTP

+---

-+--

--+-

---+

++--

+-+-

+--+

-++-

-+-+

--++

+++-

++-+

+-++

-+++

++++

Act

ivity

(%

of m

ax.

)

40

80

120

0

0

NaCl (mM)

50 100 150 200 250

ssDNA

ssRNA

Ladder

Act

ivity

(%

of m

ax.

)

40

80

120

030

Temperature (°C)

35 40 45 50

ssDNA

ssRNA

Ladder

D

Figure S1.

1 -

30 -

A

5

Figure S2.

A B C

dsRNA -

DNA/RNA -

QDE-1 DN

ssRNA

ssDNA

0

1

1

0.03

1

1

0.08

1

1

0.15

1

1

0.3

1

1

1

1

1

1

1

0

1

1

0.1

1

1

0.25

1

1

0.5

1

1

1

1

0

1

1

0.1

1

1

0.25

1

1

0.5

1

1

1

1

Relative concentrations:

Rela

tive a

ctiv

ity (

% o

f m

ax.

)

Relative QDE-1 conc.

120

80

40

0

20

60

100

0 0.5 1.0 1.5 2.0

DNA/RNA

dsRNA

2

1

1

Rela

tive a

ctiv

ity (

% o

f m

ax.

)

Relative ssDNA conc.0 0.4 0.6 0.8 1.0

DNA/RNA

dsRNA

0.2

Rela

tive a

ctiv

ity (

% o

f m

ax.

)

120

80

40

0

20

60

100

Relative ssRNA conc.0 0.4 0.6 0.8 1.00.2

DNA/RNA

dsRNA

120

80

40

0

20

60

100

6

Figure S3.

1

61

121

181

241

301

361 LARSE ESARSQVQVH APVVAARLRN IWPKFPKWLH EAPLAVAWEV

421 TRLFMHCKVD LEDESLGLKY DPSWSTARDV TDIWKTLYRL DAFRGKPFPE KPPNDVFVTA

481 MTGNFESKGS AVVLSAVLDY NPDNSPTAPL YLVKLKPLMF EQGCRLTRRF GPDRFFEILI

541 PSPTSTSPSV PPVVSKQPGA VEEVIQWLTM GQHSLVGRQW RAFFAKDAGY RKPLREFQLR

601 AEDPKPIIKE RVHFFAETGI TFRPDVFKTR SVVPAEEPVE QRTEFKVSQM LDWLLQLDNN

661 TWQPHLKLFS RIQLGLSKTY AIMTLEPHQI RHHKTDLLSP SGTGEVMNDG VGRMSRSVAK

721 RIRDVLGLGD VPSAVQG FG SAKGMWVIDV DDTGDEDWIE TYPSQRKWEC DFVDKHQRTL

781 EVRSVASELK SAGLNLQLLP VLEDRARDKV KMRQAIGDRL INDLQRQFSE QKHALNRPVE

841 FRQWVYESYS SRATRVSHGR VPFLAGLPDS QEETLNFLMN SGFDPKKQKY LQDIAWDLQK

901 RKCDTLKSKL NIRVGRSAYI YMIADFWGVL EENEVHVGFS SKF DEEESF TLLSDCDVLV

961 ARS AHFPSD IQRVRAVFKP ELHSLKDVII FSTKGDVPLA KKLSGG YDG MAWVCWDPE

1021 IVDGFVNAEM PLEPDLSRYL KKDKTTFKQL MASHGTGSAA KEQTTYDMIQ KSFHFALQPN

1081 FLGMCTNYKE LCYINNSVS NKPAIILSSL VGNLVDQS Q GIVFNEASWA QLRRELLGGA

1141 LSLPDPMYKS DSWLGRGEPT HIIDYLKFSI ARPAIDKELE AFHNAMKAAK DTEDGAHFWD

1201 PDLASYYTFF KEISDKSRSS ALLFTTLKNR IGEVEKEYGR LVKNKEMRDS KDPYPVRVNQ

1261 VYEKWCAITP EAMDKSGANY DSKVIRLLEL SFLADREMNT WALLRASTAF KLYYHKSPKF

1321 VWQMAGRQLA YIKAQMTSRP GEGAPALMTA FMYAGL PDK KFTKQYVARL EGDGSEYPDP

1381 EVYEVLGDDD FDGIGFTGNG DY

MNPITPRKRN SPVEEIINRL NNDYNLGLQC VADTTLTPHR RKELAESDED FGRHDKIYRA

LNFLYWRKDD SLNQAEANFF IEAKAASSNW VPKAHADPDT LPWSKEPPRA ATAGQQWALQ

TVLLEVLNRF MPPPNNTPGR TFGRTLSGPS GLSRPTSTNT KRKDEPANVT FADPPKRSLT

RSATGPPIHG AAIPLKFPDP VNTGSKRPSL ESENLNQCTK RAKGKLSDNV AAAAAPPVPI

ASALDKVPTR RHANTRDPTA TGHRRADQVD SFDTSQGTSY GSSVFSACRH NQSTTQSSFE

APPSQPREKR PVDATVFEAG HLIESPSKGR TTKSHIDNQP LSSSSQGETS FSTYYESFPS

SGGEGAIPEP SRSNG

R

R

P D D

R K

M

D1007A DADNP964A

R1091A

R738A

M1357D/C

K1119W

R944E

DPBB1

DPBB2

FLAP

DPBB1: 678-792DPBB2: 916-1018FLAP: 1025-1161

7

ssRNA ssDNA

Figure S4.

CTLQ

DE-1 D

N WT

DA

QDE-1

DN

D1007A

P964A

R1091A

R738A

M1357D

M1357C

K1119W

R944E

CTLQ

DE-1 D

N WT

DA

QDE-1

DN

D1007A

P964A

R1091A

R738A

M1357D

M1357C

K1119W

R944E

A

B

ssRNA

ssDNA

CTLQ

DE-1 D

N WT

DA

QDE-1

DN

D1007A

P964A

R1091A

R738A

M1357D

M1357C

K1119W

R944E

8

BamfordAntti P. Aalto, Minna M. Poranen, Jonathan M. Grimes, David I. Stuart and Dennis H.

Neurospora crassa Activities of the Multifunctional RNA Silencing Polymerase QDE-1 of In Vitro

doi: 10.1074/jbc.M110.139121 originally published online July 20, 20102010, 285:29367-29374.J. Biol. Chem. 

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