Interactions between DSIF (DRB sensitivity inducingfactor), NELF (negative elongation factor), and theDrosophila RNA polymerase II transcriptionelongation complexAnamika Missra and David S. Gilmour1

Center for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802

Edited by Robert G. Roeder, The Rockefeller University, New York, NY, and approved May 7, 2010 (received for review January 20, 2010)

Negative elongation factor (NELF) and 5,6-dichloro-1-β-D-ribofura-nosylbenzimidazole sensitivity-inducing factor (DSIF) are involvedinpausingRNAPolymerase II (Pol II) in thepromoter-proximal regionof the hsp70 gene in Drosophila, before heat shock induction. Suchblocks in elongation are widespread in the Drosophila genome.However, the mechanism by which DSIF and NELF participate insetting up the paused Pol II remains unclear. We analyzed theinteractions among DSIF, NELF, and a reconstituted Drosophila PolII elongationcomplex togain insight into themechanismofpausing.Our results show that DSIF and NELF require a nascent transcriptlonger than 18 nt to stably associate with the Pol II elongationcomplex. Protein-RNA cross-linking reveals that Spt5, the largestsubunit of DSIF, contacts the nascent RNA as the RNA emerges fromthe elongation complex. Taken together, these results provide apossible model by which DSIF binds the elongation complex viaassociation with the nascent transcript and subsequently recruitsNELF. Although DSIF and NELF were both required for inhibitionof transcription, we did not detect a NELF-RNA contact when thenascent transcript was between 22 and 31 nt long, which encom-passes the region where promoter-proximal pausing occurs onmany genes in Drosophila. This raises the possibility that RNAbinding by NELF is not necessary in promoter-proximal pausing.

transcriptional pausing ∣ gene expression ∣ negative elongation factors

Transcription by RNA Polymerase II (Pol II) in eukaryotes is ahighly regulated network of events that can be broadly sepa-

rated into the sequential stages of initiation, elongation, and ter-mination. Progression of Pol II through these stages is modulatedby the interplay of numerous factors. Of the various mechanismsthat regulate transcription, promoter-proximal pausing duringearly elongation has recently gained recognition as a widespreadrate-limiting step in metazoans (1, 2). Genomewide analyses haverevealed that thousands of genes in higher eukaryotes containtranscriptionally engaged Pol II concentrated at their promoters,indicating a postinitiation regulatory mechanism (3–5). In humancells, global analysis of nuclear run-on products detected pausedPol II on numerous genes (6). In Drosophila, promoter-proximalpausing was first identified on the uninduced hsp70 gene (7, 8).Permanganate genomic footprinting reveals that pausing typicallyoccurs 20–50 nt downstream of the transcription start site in alarge number of genes (9). Paused polymerase was observedin promoters of many genes controlling Drosophila development,suggesting the importance of this process in regulating geneexpression during complex developmental events (5, 10, 11).

Two protein complexes, DRB (5,6-dichloro-1-β-D-ribofurano-sylbenzimidazole) sensitivity-inducing factor (DSIF) and negativeelongation factor (NELF), are involved in promoter-proximalpausing in vivo (12) and have been shown to cooperatively represstranscription elongation in vitro (13). A widely accepted modelproposes that the kinase P-TEFb alleviates this repression byphosphorylating Pol II and DSIF (14–16). This model is sup-

ported by ChIP results showing that during heat shock induction,P-TEFb is recruited to the heat shock genes (17).

NELF is a multisubunit complex that is known to regulate tran-scription of many genes. For example, it was found to attenuatetranscription of some genes induced by the estrogen receptor(18). NELF is also involved in pausing Pol II on the HIV provirus,and depletion of NELF causes loss of paused Pol II and increasedvirus production (19). RNAi mediated knockdown of NELF wasshown to cause a significant reduction in the expression of numer-ous Drosophila genes that had paused Pol II, and it was proposedthat the paused Pol II prevented nucleosomes from assemblingover the core promoter region (4).

NELF is thought to inhibit elongation by binding the nascenttranscript when it exits the Pol II, thereby restricting further extru-sion of RNA (12). The NELF-E subunit has an RNA recognitionmotif (RRM), which binds RNA, and mutation of the RRMrenders NELF unable to repress elongation in vitro (16). Anothermodel, based on the sequence similarity betweenNELF-A and thehepatitis delta virus antigen posits that the NELF-A subunitassociates with the clamp domain of Pol II (20, 21). This associa-tion could alter the active site of the Pol II in a way that inhibitselongation (1).

DSIF was initially discovered as a factor that rendered Pol IItranscription sensitive to DRB, a nucleoside analog (22). DSIF iscomposed of two subunits called Spt4 and Spt5. DRB inhibitsP-TEFb, causing transcription to be sensitive to pausing by DSIFand NELF. DSIF colocalizes with Pol II at numerous loci onDrosophila polytene chromosomes (12, 23), and ChIP analysesshow that DSIF is distributed across the body of transcribed genes(23, 24). Thus, DSIF is viewed as a transcription elongation factorwith both positive and negative properties. In the context ofpromoter-proximal pausing, it is thought to stabilize the PolII-NELF interaction (16). However, some studies have indicatedthat DSIF may play a more significant role in pausing. On thehuman A20 gene, pausing of Pol II at the promoter was reportedto involve DSIF, but not NELF (25). In Caenorhabditis elegans,which has DSIF, but lacks NELF, promoter-proximal pausingoccurs in response to starvation (26).

In this study, we analyzed the interactions of Drosophila DSIFand NELF with the Pol II elongation complex, using a purified invitro system (27). We found that NELF associates with the Pol IIelongation complex only in the presence of DSIF. Surprisingly, wediscovered that DSIF, not NELF, contacts the nascent RNA as it

Author contributions: A.M. and D.S.G. designed research; A.M. and D.S.G. performedresearch; A.M. and D.S.G. analyzed data; and A.M. and D.S.G. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. E-mail: dsg11@psu.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1000681107/-/DCSupplemental.

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emerges from Pol II. This DSIF-RNA contact could be a decisivestep in pausing the polymerase.

ResultsPurification of Active FLAG NELF Complexes. The NELF complexconsists of four subunits: NELF-A, NELF-B, NELF-D, andNELF-E. To purify theDrosophilaNELF complex, wemade trans-genic flies that expressed FLAG-tagged versions of the B, D, or Esubunits. Nuclear extracts were prepared from embryos andsubjected to immunoaffinity purification using anti-FLAG beads.All four subunits of NELF were detected upon analysis of thepurified samples on aCoomassie-stained, SDS-polyacrylamide gel(Fig. 1A, lanes 2–4). Comparison of these protein samples to amock purified protein sample from non-FLAG expressing Droso-phila nuclear extract (Fig. 1A, lane 1) revealed some proteins thatbind the FLAG column nonspecifically (marked with asterisks),and also some that appeared to copurify with the NELF complex.The identities of these NELF-associated proteins are being inves-tigated. The FLAG NELF-D complex showed the best yield so itwas used in the following experiments. Western blot analysisof the FLAG NELF-D complex detected all four NELF subunits(Fig. 1B).

To evaluate the activity of our NELF and DSIF preparations,we set up an in vitro elongation system (Fig. 2A) using a tailedtemplate and purified Drosophila Pol II (Fig. 1C), as describedpreviously (27). As outlined in Fig. 2B, Pol II was allowed totranscribe to the end of a 22-nt-long G-less cassette and stalledby the absence of GTP. The stalled elongation complexes were

incubated with buffer, purified DSIF (Fig. 1C), NELF, or bothproteins together. GTP was then added to the reaction to resumeelongation beyond the G-less cassette, and transcript lengthswere monitored at different times. As shown in Fig. 2C, addingDSIF or NELFalone had no effect on the elongation rate relativeto buffer alone (compare lanes 2–5 with lanes 10–13 or 14–17). Incontrast, addition of both DSIFand NELF inhibited transcriptionand caused appearance of shorter transcripts at all the time points(lanes 6–9). These results indicate that DSIFand NELFare activeand are both required for transcription inhibition.

DSIF and NELFAssociate Stably with the Pol II Elongation Complex.Weused a native gel-shift assay to analyze the association of DSIFand NELF with the Pol II elongation complex. A complex con-taining a 70-nt-long radioactive transcript, template DNA, andPol II (EC70) was visualized on a native gel (Fig. 3A, lane 1).Addition of DSIF caused a concentration-dependent shift inthe migration of the Pol II complex (lanes 2–4), while additionof NELF did not (lanes 5–7), indicating that DSIF can bindthe Pol II elongation complex alone, but NELF cannot. Whenboth DSIFand NELF were added to EC70, the complex migratedslower than the DSIF-bound complex, indicating that the bindingof NELF requires DSIF (lane 11). The binding of DSIF andNELF appears to be cooperative as formation of a complex con-taining both proteins is evident at a low amount of DSIF that failsto stably associate with the elongation complex by itself (Fig. 3B).This demonstrates clearly that DSIF and NELF associatestably with a Pol II elongation complex.

To verify that the slower migrating complex contained Pol II,DSIF, and NELF, we added antibodies for each of these proteinsand tested if the resulting complex was supershifted. Addingpreimmune serum did not affect migration of the complex

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Fig. 1. Purification of Drosophila NELF. (A) Nuclear extracts were preparedfrom transgenic fly embryos expressing FLAG-NELF subunits and fractionatedusing anti-FLAG Sepharose (Sigma). Eluates (10 uL) were analyzed by SDS-PAGE and Coomassie staining (lanes 2–4). Arrows indicate positions of theNELF subunits. Lane 1 (Control) shows 10uL of eluate fromamock purificationofnuclear extract fromnontransgenic embryos. Asterisks denoteproteins thatbind nonspecifically to the FLAG column. (B)Western blot analysis of the FLAGNELF-D complex using NELF-A, NELF-B, NELF-D, and NELF-E antibodies. (C) Sil-ver-stained gel with Pol II purified from Drosophila embryo nuclear extractand Coomassie blue-stained gel with DSIF purified from baculovirus. Theprominent band migrating just above Rpb3 is an unidentified contaminant.

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Fig. 2. NELF and DSIF inhibit transcription elongation. (A) Schematic repre-sentation of the in vitro elongation system. RNA Pol II initiates transcriptionwith UpG on the tailed template and stalls before the four Gs located at theend of the G-less cassette when GTP is absent. Upon addition of GTP, Pol II isable to resume elongation and generate a runoff transcript. (B) Outline ofthe experiment to measure the elongation rate of Pol II in the presenceor absence of DSIF and NELF. (C) Analysis of elongation rates. Prior to additionof GTP, the stalled elongation complexes were incubated with buffer (lanes2–5), DSIF (lanes 10–13), NELF (lanes 14–17), or DSIF and NELF (lanes 6–9).Lane 1 shows transcripts isolated from the stalled elongation complex beforeaddition of proteins and GTP.

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(Fig. S1, lane 3), but addition of antibodies against Pol II (Rpb3),DSIF (Spt5), NELF-D, and FLAG resulted in supershifted com-plexes (Fig. S1, lanes 4, 7–10), indicating that NELF and DSIFbound to the Pol II elongation complex.

Binding of DSIF Depends on the Length of the Nascent Transcript.Since one model by which NELF and DSIF inhibit elongationposits that NELF associates with the nascent transcript (12),we wanted to determine if the length of the nascent transcriptinfluences binding of these proteins. We modified the lengthof the G-less cassette in our tailed template, to generate 31,27, 22, 18, or 14-nt-long nascent transcripts (Fig. 4 A and B).DSIF alone or in combination with NELF bound to elongationcomplexes with a 22-nt-long RNA (Fig. 4C, lanes 1–4) but showedalmost no binding to a complex with an 18- or a 14-nt-long RNA(lanes 5–12). Binding of DSIF and NELF was also observed toEC31 and EC27 (Fig. S2). These results show that the transcriptmust be longer than 18 nt for these proteins to bind. Since theRNA is about 18 nt long when it begins to emerge from the bodyof the polymerase (28), our results suggest that DSIF and NELFbind to the newly emerged RNA.

DSIF Cross-Links to the Nascent Transcript. Since DSIF alone exhib-ited binding to EC22 (Fig. 4C, lane 2), it was possible that DSIFcontacted the nascent transcript as it emerged from the elongationcomplex. To test this, we performed protein-RNA cross-linkingstudies on the elongation complexes that were bound to DSIFand NELF. Transcription reactions were assembled as before on

the tailed template, except that theUV cross-linkable UTPanalog5-bromo UTP and 32P-CTP were used. When DSIF and NELFwere absent, we detected two bands corresponding to Rpb1 andRpb2 cross-linked to the RNA in the EC31 complex (Fig. 5A, lane2).WhenDSIFandNELFwere present, wedetected another bandcorresponding to the size of the Spt5 subunit of DSIF (lane 4; Spt5migrates slightly slower than Rpb2). Similar results were obtainedwith the EC27 and EC22 complexes (Fig. S3). In contrast, theRNA in EC18 did not cross-link to Spt5, although Rpb1 andRpb2 cross-linking were seen (lanes 6 and 8). None of the NELFsubunits were seen to cross-link to any of these complexes. Pre-viously reported UV cross-linking studies have shown that thenascent RNA contacts the Rpb7 subunit of human Pol II whenit is between 26–32 nt long (29). The predicted molecular weightof Drosophila Rpb7 is approximately 19 kDa, and we detected afaint band in this region of the gel in the EC31 complex but notthe EC18 complex (Fig 5A, lane 2, asterisk).

The cross-linked EC31 complex was immunoprecipitated withantibodies against Pol II, Spt5, or NELF-E to verify the identityof the cross-linked bands (Fig. 5B). Immunoprecipitations werecarried out under conditions that would disrupt protein-proteininteractions (details in Materials and Methods). In the input(lane 1), bands of expected mobility corresponding to Rpb1,Rpb2, and Spt5 were detected. The Rpb1 and Spt5 bands wererecovered after immunoprecipitation with the respective antibo-dies (lanes 2 and 3). No bands corresponding to NELF-E wereseen in the input or immunoprecipitated lanes (lanes 1 and 4).These results confirm that the Spt5 subunit of DSIF contactsthe nascent transcript.

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Fig. 3. DSIF and NELF form a stable complex with the Pol II elongation com-plex. (A) Native gel analysis of elongation complexes. Lane 1 shows a complexof Pol II with a 70-nt radiolabeled nascent transcript and the tailed template(EC70). Approximately 100 ng (∼0.25 pmol) of Pol II was present in each sam-ple. Elongation complexes stalled at the end of the G-less cassette wereincubated with increasing amounts of purified DSIF (approximately 0.5 pmol,1 pmol, and 2 pmol of DSIF; lanes 2–4) or purified NELF (approximately0.15 pmol, 0.3 pmol, and 0.6 pmol of NELF; lanes 5–7). Lanes 8 and 9 showelongation complexes incubatedwith 0.5 pmol of DSIF and 0.15 pmol of NELF,respectively, and lane 10 shows a complex with both proteins added (0.5 pmolofDSIF and 0.15 pmol of NELF). The concentrations of all proteinswere judgedby comparing intensities of their bands to that of known amounts of proteinmarkers on a Coomassie-stained gel. Therefore, these concentrations are onlyestimates and merely indicate that none of the proteins are present in largeexcess over the others. (B) Evidence for cooperative binding by DSIF and NELF.Approximately 100 ng (∼0.25 pmol) of Pol II was present in each sample. Lane1 shows EC70 alone. Lane 2–5 have 0.25, 0.5, 0.75, and 1.0 pmol of DSIF and noNELF. Lanes 6–9 have 0.05, 0.1, 0.15, and 0.2 pmol NELF and 0.25 pmol of DSIF.Lanes 10–13 have 0.05, 0.1, 0.15, and 0.2 pmol NELF and 0.5 pmol of DSIF.

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Fig. 4. Binding of DSIF and NELF depends on the length of the RNA.(A) Schematic showing elongation complexes with different lengths of thenascent RNA generated by modifying the length of the G-less cassette.(B) Analysis of RNA produced by transcription on templates containing70-, 31-, 27-, 22-, 18-, or 14-nt-long G-less cassettes on a denaturing gel.(C) Binding of DSIF and NELF to elongation complexes with different lengthsof nascent transcripts. Elongation complexes containing 22-, 18-, or 14-nt-longnascent transcriptswere analyzed on a native gel (lanes 1, 5, and 9). Each com-plexwas incubatedwith 1 pmol of DSIF (lanes 2, 6, and 10) or 0.3 pmol of NELF(lanes 3, 7, and 11) or 1 pmol of DSIF and 0.3 pmol of NELF (lanes 4, 8, and 12).

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Both DSIF and NELF Contact the RNA in EC70. The human NELF-Esubunit has been shown to have RNA binding activity (16). Sincewe did not detect NELF-E or any other NELF subunits cross-linked to the nascent transcript that was 22–31 nt long, we testedif a longer RNA allowed binding to NELF. Adding NELF to anEC70-DSIF complex resulted in cross-linking of a polypeptideof the size of NELF-E (Fig. 6A, compare lanes 1 and 2). WhenNELF was added alone, no band corresponding to NELF-Ewas observed, indicating that free NELF did not bind nonspecifi-cally to the RNA (Fig. S4, compare lanes 2 and 4). Immunopre-cipitation usingNELF-E antibody verified the identity of NELF-E(Fig. 6B, lane 4 and Inset). These results suggest that NELFcontacts the nascent transcript farther from the 3′ end than DSIF.

DiscussionDSIFand NELFare key factors in pausing Pol II in the promoter-proximal region of genes in Drosophila and human cells (12, 16,22). To gain insight into the mechanism by which DSIFand NELFcontribute to promoter-proximal pausing, we developed a systemin which we could monitor the physical interaction of DSIF andNELF with a Pol II elongation complex using a native gel elec-trophoresis assay. Previously, we had demonstrated that DSIFalone could associate with the Pol II elongation complex (27).Here, we have developed a method to purify Drosophila NELF,thus allowing us to explore the interplay of DSIF and NELF withthe elongation complex.

DSIF and NELF Bind Cooperatively to the Pol II Elongation Complex.Our results show that the association of NELFwith the elongation

complex is dependent on the presence of DSIF (Figs. 3A and 4C).Previous work provided evidence that NELF associated with pre-formed complexes of DSIF and Pol II in nuclear extracts but theinteraction of DSIF and Pol II was not dependent on NELF (16).These interactions were likely occurring outside the context of anelongation complex and were relatively weak because the bulk ofDSIF, NELF, and Pol II exist independent of each other in nuclearextracts. In contrast, our results show that NELF can significantlyinfluence the binding of DSIF to Pol II within the context of anelongation complex when limiting amounts of DSIF are present(Figs. 3A and B and 4C). Since Pol II, DSIF, and NELF have beenshown to interact individually with each other (16, 30), it is likelythat this network of interactions contributes to stable associationof these proteins in the context of the elongation complex.

The Nascent Transcript Significantly Impacts Binding of DSIF and NELF.Our binding assays show that the length of the nascent transcriptaffects the association of DSIF and NELF with the elongationcomplex. While binding of DSIF alone or in combination withNELF to the elongation complex was evident for an elongationcomplex with a nascent transcript of 22 nt, no binding wasdetected when the nascent transcript was 18 nt long (Fig. 4C).These results are consistent with the finding that human DSIFand NELF require transcripts ≥18 nt long to inhibit transcription(31), and also a recent study showed human DSIF preferentiallybound elongation complexes containing transcripts that were atleast 25 nt long (32). The 5′ end of an 18-nt-long nascent tran-script just begins to emerge from the surface of Pol II (28, 33).Exposure of four additional nucleotides appears to be sufficientfor binding of DSIF alone or with NELF. Notably, the associationof DSIF with the elongation complex is not simply due to non-specific interaction with the RNA or DNA because previousexperiments show that binding of DSIF to the elongation com-plex requires specific contacts with Pol II (27).

One way in which nascent transcript length could affect theassociation of DSIFand NELF is by providing an additional bind-ing site in the elongation complex. Previous results have directedattention at an RRM in NELF-E (16). Mutations in this RRMimpair the capacity of NELF to inhibit elongation in the presenceof DSIF. However, these experiments focused on elongation overdistances greater than 100 nt. Our finding that DSIF associateswith EC22 but not EC18 suggests that DSIF rather than NELFmight be interacting with the nascent transcript, and our RNA-protein cross-linking data support this hypothesis. The Spt5

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Fig. 5. Spt5 contacts the nascent RNA. (A) Results of UV cross-linking of tran-scription complexes EC31 and EC18. The complexes were left untreated (lanes2 and 6) or incubated with DSIF and NELF (lanes 4 and 8) before cross-linking.Samples were then treated with RNase A and DNase I, precipitated withtrichloroacetic acid, and subjected to SDS-PAGE on a 4%–20% gradient gel(Bio-Rad). Lanes 1, 3, 5, and 7 represent non-cross-linked samples. The bandmarked with the asterisk may be Rpb7 (see text). (B) Immunoprecipitationof the cross-linked EC31 complex bound toDSIF andNELF. The antibodies usedare shown above each lane. The Rpb1 antibody, 8WG16, associates with theCTD of Rpb1. Forty percent of the inputs were loaded in lane 1. The sampleswere analyzed by 8% SDS-PAGE. The sequence of the nascent transcript inEC31 and EC18 is shown at the bottom. The cross-linkable Us are underlined,and the radioactive Cs are marked with asterisks.

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Fig. 6. NELF-E contacts the RNA in EC70. (A) UV cross-linking of transcriptioncomplex EC70 bound to DSIF only (lane 1) or DSIF and NELF (lane 2). (B) Im-munoprecipitation of EC70 by different antibodies. (Inset) Part of the gelwithenhanced contrast to display the NELF-E bandmore clearly. Image processingwas done using the curves tool in Adobe Photoshop. The sequence of thenascent transcript in EC70 is shown and annotated as described in Fig. 5.

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subunit of Drosophila and human DSIF contains five Kyprides,Ouzounis,Woese (KOW)domains.An isolatedKOWdomain fromAquifex aeolicusNusGhas been shown to associate withRNA(34),so it is possible that one of these domains in Spt5 is contacting thenascent transcript as it emerges from the elongation complex.

Our cross-linking analysis detected contact between NELF-Eand the nascent transcript in EC70 (Fig. 6) but not in EC31(Fig. 5). The 5′ end of the nascent transcript contacts Rpb7 whenits length is between 26 to 32 nt (29). Therefore it is possible thata longer nascent transcript is required to allow contact withNELF. Given that promoter-proximal pausing can occur beforePol II transcribes 30 nt (35), we propose that the RRM of NELF-E is not involved in promoter-proximal pausing. Its role could belimited to processes involving longer nascent transcripts such asregulation that appears to involve the transactivation responseelement of HIV (36) or 3′ end formation of histone mRNAs (37).

Insights into Promoter-Proximal Pausing. The finding that DSIF andNELF associate with EC22 but not with EC18 is very relevant tothe process of promoter-proximal pausing. Permanganate geno-mic footprinting of over 60 different promoters reveals that Pol IIpauses in the promoter-proximal region 20 to 50 nt downstreamfrom the transcription start site (9). Those cases where the Pol IIappeared to be pausing closer to a transcription start site werefound to have the start sites inaccurately mapped (38). Thus,the promoter-proximal limit for the range where Pol II pausesis likely to be dictated by the minimum length of RNA requiredfor DSIF to associate with the elongation complex.

From the results presented here, we propose that the first stepin promoter-proximal pausing involves binding of DSIF to thenascent transcript. NELF subsequently associates to form a stablecomplex. Importantly, this complex alone is not sufficient to sta-bly pause the Pol II as our results (Fig. 2C) and those of others(15) show that elongation is slowed but not halted in reactionsinvolving only these three proteins. Hence other factors thatremain to be identified are likely to act in concert with this corecomplex of DSIF, NELF, and Pol II to stably pause Pol II in thepromoter-proximal region of genes. Since transcription in vivooccurs on chromatin, nucleosomes may cooperate with DSIFand NELF in setting up the paused polymerase (39, 40). Theexperimental approach described here could serve as a way toidentify additional factors involved in pausing.

Materials and MethodsTransgenic Flies. cDNA sequences of NELF-B, NELF-D, or NELF-E were amplifiedfrom the plasmids pRMHA3.FLAG NELF-D, pRMHA3.FLAG NELF-B andpA5cΔP.FLAG NELF-E and inserted downstream of the hsp83 promoter inpCaSpeR-hs83 (41). The resulting plasmids encoded NELF-D and NELF-Bwith two C-terminal FLAG tags and NELF-E with a single N-terminal FLAGtag. Drosophila transformation was done by standard methods (RainbowTransgenic Flies, Inc.).

Purification of NELF and DSIF. Large populations of transgenic flies expressingFLAG-tagged NELF-B, NELF-D, or NELF-E were used for making nuclearextracts. Drosophila nuclear extracts were prepared from approximately40 g of embryos as described previously (42) and dialyzed against 100 mMKCl-HEMG (25 mM Hepes pH 7.6, 12.5 mM MgCl2, 0.1 mM EDTA pH 8,10% glycerol), supplemented with 1 mM DTT, 0.5 mM sodium bisulfite,and 0.1 mM PMSF until the conductivity of the extract matched that of180 mM KCl-HEMG. The extract (8 mL at ∼24 mg∕mL protein) was incubatedwith 300 μL of anti-FLAG beads (Sigma) for 4 h at 4 °C. The beads werewashed with 5 mL of 180 mM KCl-HEMG, three times for 10 min each andthen with 5 mL of 100 mM KCl-HEMG, two times for 5 min each at 4 °C. FLAGNELF complexes were eluted with 300 μL of 100 mM KCl-HEMG containingFLAG peptide (100 ng∕μL, Sigma) for 30 min at 4 °C. Purification of DSIF isdescribed in ref. 27. This involves tandem FLAG and HA-tag affinity purifica-tions of coexpressed FLAG-Spt5 and HA-Spt4 from baculovirus-infected cells.Spt5 in this preparation can be phosphorylated with P-TEFb and methylatedwith PRMT1 indicating that some portions of these potential modificationsites in Spt5 are unmodified.

Purification of RNA Pol II from Drosophila Embryos. Purification of RNA Pol IIwas based upon a previously described protocol (43), which was used becausethe carboxyl-terminal domain (CTD) of the largest subunit of Pol II remainedintact during purification. Five 10-g portions of 0- to 12-h embryos, which hadbeen previously dechorionated and stored at −80 °C, were processed in suc-cession. Each portion was homogenenized in 40 mL of 0.3 M HGAED/pc with10 strokes in a motorized dounce homogenizer with a Teflon pestle. HGAED/pc corresponds to 25 mM Hepes pH 7.6, 15% glycerol, 0.1 mM EDTA, 1 mMDTT, 1 mM PMSF, 16 μg∕mL Benzamidine HCl, 10 μg∕mL aprotinin, 10 μg∕mLPepstatin A, 10 μg∕mL Leupeptin, 5 μg∕mL soybean trypsin inhibitor, and themolar concentration that precedes this refers to the concentration of ammo-nium sulfate. DTTand all protease inhibitors were added fresh. Homogenatesfrom 10-gramportionswere filtered through one layer ofMiracloth. The totalhomogenate from 50 g of embryos was distributed equally among eight OakRidge ultracentrifuge tubes and centrifuged at 45,000 rpm for 1 h at 4 °C in a70Ti rotor. The clear liquid located between a topwhite layer and a gelatinouspellet was collectedwith a large bore syringe. This yielded∼165 mLof extract.

The lysate was diluted with an equal volume of 25 mM Hepes pH 7.6, 15%glycerol, and 0.1 mM EDTA and loaded at 5 mL∕min onto a 300-mL DEAEcellulose column previously equilibrated with 0.12 M HGAED∕0.1 pc(0.1 pc indicates that the concentration of protease inhibitors was reducedtenfold). The column was washed with 900 mL of 0.12 M HGAED∕0.1 pc, andproteins were eluted with a 1,400 mL 0.12 to 0.45 M HGAED∕0.1 pc gradient.Pol II activity was assayed as described (43) and observed to elute in twodistinct peaks. The second peak of Pol II activity, which had the highestspecific activity, was diluted with 0 M HGAED∕0.1 pc to a conductivity thatmatched 0.2MHGAED and then loaded onto an 8-mL POROS heparin columnpreviously equilibrated with 0.2 M HGAED∕0.1 pc. The column was washedwith 20 mL of 0.2 M HGAED∕0.1 pc, and Pol II was eluted with a 40-mLgradient from 0.2 to 0.6 M HGAED∕0.1 pc at a flow rate of 2 mL∕min.The peak of Pol II activity was diluted with 0 M HGAED∕0.1 pc to a conduc-tivity that matched 0.2 M HGAED and was loaded onto a Mono Q 10∕10column previously equilibrated with 0.2 M HGKED∕0.1 pc (HGKED containspotassium chloride instead of ammonium sulfate). The column was washedwith 30 mL of 0.35 M HGKED∕0.1 pc, and Pol II was eluted with an 80-mLgradient from 0.35 to 0.6 M HGKED∕0.1 pc. SDS-PAGE analysis of the PolII indicated that CTD of the largest subunit of Pol II was intact.

DNA Templates. Templates were PCR amplified from a plasmid containing aG-less cassette. The forward primer included a BglII site and was modified toamplify different lengths of the G-less cassette. The same reverse primer wasused for generating all lengths of template. The PCR products had a 70-, 31-,27-, 22-, 18-, or 14-nt-long G-less cassette followed by 80-nt DNA sequencecontaining all four nucleotides. These were digested with BglII [New EnglandBiolabs (NEB)], dephosphorylated with Antarctic phosphatase (NEB), andligated to a 5′-phosphorylated oligonucleotide with the sequence GAT-CAAAAAAAATTA. The resulting 11-nt overhang served as an initiation sitefor Pol II in the presence of the starting dinucleotide UpG (Sigma).

Generation of Stalled Elongation Complexes. Transcription reactions (15 μL)contained 50 mM Hepes pH 7.6, 200 mM KCl, 1 mM MnCl2, 12% glycerol,0.5 mM DTT, 0.5 mM UpG, 20 units of RNasin (Promega), 100 ng of template,and ∼100 ng of purified Drosophila Pol II. The template was preincubatedwith Pol II for 5 min in the transcription buffer, and then transcriptionwas initiated by adding a 5 μL NTP mix, yielding final concentrations of0.1 mM ATP, 0.1 mM CTP, 5 μM UTP, 5 μM 3′O-methyl GTP, and1 μCi∕reaction of ½α-32P� UTP. Each reaction was incubated at 21 °C for 25 min.

Binding of Proteins to the Elongation Complexes. Purified DSIF and NELF wereadded to the transcription reactions and allowed to bind to the Pol II elonga-tion complex for 15 min. Five micrograms of yeast RNAwas then added to thereactions to reduce nonspecific binding of proteins to the nascent transcript.In cases where antibodies against different proteins were used, the antibo-dies were added to the reactions after the 15-min incubation with theproteins and further incubated for 10 min. The samples were analyzed onnative gels, processed for UV cross-linking, or used for RNA extraction.

Analyses of Elongation Complexes by Native Gel Electrophoresis. Gel runningbuffer (RB) contained50mMTris pH8.5, 0.38MGlycine, 2mMEDTA, and5mMMgCl2. Sixty milliliters of the 4% acrylamide gel mix were made using 6 mL of40∶1 acrylamide:bis solution, 12 mL of 5X RB, 1.5 mL of 100% glycerol, and30 μL of 1 M DTT. One hundred fifty milliliters of 25% ammonium persulfateand 55 μL of N,N,N′,N′-tetramethylethylenediamine were used to polymerizethe gel mix overnight. Gels (20 cm × 20 cm × 1.5 mm) were prerun at 100 Vat4 °C for 90 min. Samples were loaded onto the gels without the addition of

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dye. A Bromophenol Blue-Xylene Cyanol dye mix was loaded in an adjacentlane to track the progress of the electrophoresis. Gels were run at 200V for 4 hat 4 °C, transferred to paper, dried, and analyzed with a PhosphorImager.

Analyses of Transcripts. Transcription reactions were stopped with 40-μL stopbuffer (20 mM EDTA pH 8.0, 0.2 M NaCl, 1% SDS, 0.25 mg∕mL yeast RNA,0.1 mg∕mL proteinase K). Samples were incubated at room temperaturefor 10 min and then extracted once with 60 μL of phenol/chloroform/isoamylalcoholmixture (25∶24∶1). TheRNAwas ethanol precipitated andanalyzedonan 8% denaturing gel. The gels were dried on paper and analyzed using aPhosphorImager.

Protein-RNA UV Cross-Linking. In vitro transcription reactions were carried outusing 0.1 mM ATP, 0.1 mM Bromo-UTP, 5 μM CTP, 5 μM 3′O-methyl GTP, and1 μCi∕reaction of ½α-32P� CTP. Protein-bound complexes were UV cross-linkedon ice for 10 min, using a 300-nm wavelength lamp. The samples were thentreated with DNase I (1 unit) and RNase A (1 μg) for 15 min at room tempera-ture to digest the template and RNA that was not bound to protein. Thisresults in tagging of proteins with a small piece of radioactive RNA. Theprotein-RNA complexes were trichloroacetic acid precipitated or immunopre-cipitated and analyzed by SDS-PAGE.

Immunoprecipitation of Cross-Linked Complexes. Cross-linked samples weretreated with DNase I and RNase A and then supplemented with SDS andDTT to final concentrations of 1% and 10 mM, respectively. The samples wereheated to 55 °C for 10min. Immunoprecipitation (IP) buffer (50mMHepes pH7.5, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate and protease

inhibitor cocktail)was added to the reactions such that the final concentrationof SDS in the samples was reduced to 0.1%. Antibodies (8WG16, Spt5 orNELF-E) were then added to the samples and incubated overnight at 4 °C, fol-lowedby incubationwith Protein A Sepharose beads (GE) for 4 h at 4 °C. Beadswere washed three times for 10 min each with IP buffer at 4 °C. Samples wereboiled for 4 min in 1X SDS sample buffer and analyzed by SDS-PAGE.

Transcription Elongation Analyses. Stalled elongation complexes were gener-ated as described above, with the following modifications. Each reaction wasscaled up to 60 μL and contained 10 μMATP and CTP and 1 μMUTP. O-methylGTP was excluded, so that transcription beyond the G-less cassette could beresumed upon addition of GTP. After 25 min of transcription to generatestalled elongation complexes, MgCl2 was added to a final concentrationof 3 mM. We found that the presence of Mg2þ during the elongation reac-tion increased the sensitivity of transcription to inhibition by DSIF and NELFpossibly because it increases the nucleotide specificity of the Pol II (44). Thereactions were then incubated with buffer or protein for 15 min. Ten-micro-liter samples were transferred to tubes containing 40 μL of stop buffer beforeand after incubation with buffer or proteins (corresponding to −150 and 0′time points). Finally, GTP was added to the remaining reaction to a finalconcentration of 10 μM, and 10-μL aliquots were removed 2, 4, and 8 minafter the addition of GTP. RNA was extracted from all the samples, andtranscripts were analyzed on an 8% denaturing gel.

ACKNOWLEDGMENTS. We thank Dr. Chwen-Huey Wu for FLAG-DSIF andDr. Joseph Reese for helpful discussions. This work was supported by NationalInstitutes of Health Grant GM047477 to D.S.G.

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