Amanitin Greatly Reduces the Rate of Transcription by RNAPolymerase II Ternary Complexes but Fails to Inhibit SomeTranscript Cleavage Modes*

(Received for publication, June 6, 1996)

Michael D. Rudd‡ and Donal S. Luse§

From the Department of Molecular Biology, Cleveland Clinic Foundation Research Institute, Cleveland, Ohio 44195 and‡Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine,Cincinnati, Ohio 45267-0524

The toxin a-amanitin is frequently employed to com-pletely block RNA synthesis by RNA polymerase II. How-ever, we find that polymerase II ternary transcriptioncomplexes stalled by the absence of NTPs resume RNAsynthesis when NTPs and amanitin are added. Chainelongation with amanitin can continue for hours at ap-proximately 1% of the normal rate. Amanitin alsogreatly slows pyrophosphorolysis by elongation-compe-tent complexes. Complexes which are arrested (that is,which have paused in transcription for long periods inthe presence of excess NTPs) are essentially incapable ofresuming transcription in the presence of a-amanitin.Complexes traversing sequences that can provoke ar-rest are much more likely to stop transcription in thepresence of the toxin. The substitution of IMP for GMPat the 3* end of the nascent RNA greatly increases thesensitivity of stalled transcription complexes to aman-itin. Neither arrested nor stalled complexes display de-tectable SII-mediated transcript cleavage followingamanitin treatment. However, arrested complexes pos-sess a low level, intrinsic transcript cleavage activitywhich is completely amanitin-resistant; furthermore,pyrophosphorolytic transcript cleavage in arrestedcomplexes is not affected by amanitin.

The mushroom toxin a-amanitin, a bicyclic octapeptide, haslong been used as a specific inhibitor of RNA polymerase II(1–3). Calf thymus polymerase II has been shown to binda-amanitin very tightly with a stoichiometry of 1:1, aKd of 10

29

M and a complex-dissociation half-time of about 100 h at 0 °C(4). Using genetic and biochemical techniques, the amanitinbinding site has been localized to the largest subunit of RNApolymerase II (5, 6). While the mechanism of amanitin’s actionhas not been demonstrated in detail, it is known that amanitin-blocked transcription complexes can resume RNA synthesisafter irradiation with 314-nm light, which selectively destroysthe toxin (7). Thus, it seems unlikely that amanitin acts bypermanently disabling part of the polymerase, for example bycleaving one of the subunits. It has also been shown that thetoxin does not change affinity of the polymerase for nucleotides(4).

Both our laboratory (8, 9) and others (10) had observed thatpromoter-initiated RNA polymerase II ternary elongation com-plexes can form one or more phosphodiester bonds after aman-itin treatment. The combination of these results and the recentfinding that the RNA polymerase II ternary complex can cat-alyze phosphodiester bond cleavage as well as bond formation(11, 12) prompted us to perform a detailed reinvestigation ofthe effects of amanitin on RNA polymerase II elongation com-plexes. We report here that RNA polymerase II ternary com-plexes are generally able to continue RNA synthesis in thepresence of a-amanitin, albeit at greatly reduced rates. Inter-estingly, both intrinsic cleavage activity and pyrophosphoro-lytic cleavage are completely amanitin resistant in arrestedcomplexes. Given the possibility that arrest may result from aretreat of the active site of RNA polymerase away from the 39end of the nascent RNA (13), these observations suggest thata-amanitin inhibits RNA polymerase II by disrupting the in-teraction of the enzyme with the 39 end of the nascent tran-script. Our findings also lend further support to a model oftranscriptional arrest in which an equilibrium exists betweencatalytically active and inactive states.

MATERIALS AND METHODS

Ribonucleoside triphosphates were obtained from Pharmacia BiotechInc., except for ITP which was purchased from Sigma. We used ultra-pure (fast protein liquid chromatography-purified) NTPs for transcrip-tion reactions with preinitiation complexes and standard purity NTPsfor chase reactions. Labeled ribonucleotides, either [a-32P]CTP or[a-32P]UTP at 800 Ci/mmol, were purchased from DuPont NEN, Bio-Gel A1.5 m was acquired from Bio-Rad, and a-amanitin was purchasedeither from Boehringer Mannheim or Sigma.Plasmids—All plasmids used in this study were based on pML5A,

which contains the adenovirus 2 major late promoter cloned intopUC18. Plasmids pML5A (14), pML5–4NR (15), and pML20-U158 andpML20-U160 (16) have been described in detail. The pML20-U158plasmid was referred to as pML20-G155 in Izban and Luse (16); theconstruction of the pML20 precursor for pML20-U158/U160 was de-scribed in Izban and Luse (11). We constructed pML5-MUT3 frompML5A by substituting a synthesized fragment having the sequence59-GATCCTTTTTTCTCCATTTTA (nontemplate strand) for the 30-nt1

BamHI-HindIII fragment which begins at 139 downstream of tran-scription start. The pML16 series plasmids were all built from a com-mon precursor, pML16LNK, which was derived from pML5A by replac-ing a BssHII-BamHI fragment, spanning from 213 to 138 relative tothe major late promoter, with a synthesized oligonucleotide. The syn-thesized fragment bore the original sequence between 213 and 115 butchanged the remaining nontemplate strand sequence from 59-GCT-GTCTGCGTGGGCCTGCTAAG to 59-CCTTTCCCGGGCGAGCTCGG-GCCCTTG. The new sequence contains unique XmaI and ApaI sites.The pML16220 template was assembled from pML16LNK by replacingthe XmaI-ApaI segment with a 228-nt XmaI- ApaI fragment containingthe U-free cassette from pGR220 (17) (a gift from C. Kane). Thus, the

* This work was supported by National Institutes of Health GrantGM29487. The costs of publication of this article were defrayed in partby the payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734solely to indicate this fact.§ To whom correspondence should be addressed: Dept. of Molecular

Biology, NC20, Cleveland Clinic Foundation Research Institute, 9500Euclid Ave., Cleveland, OH 44195. Tel.: 216-445-7688; Fax: 216-444-0512; E-mail: lused@cesmtp.ccf.org. 1 The abbreviation used is: nt, nucleotide(s).

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 35, Issue of August 30, pp. 21549–21558, 1996© 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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nontemplate strand of pML16220 has no G residues from 11 to 123and no U residues for the next 135 bases. The pML16C27 andpML16T27 plasmids were built by inserting modified XmaI/ApaI frag-ments from pGR220 into pML16LNK. These fragments were generatedusing the polymerase chain reaction. The 39-end primer, 59-GGGAA-CAAAAGCTGGGTACCGGGCCC, overlapped the ApaI site (under-lined) and extended 25 nt downstream into the parent vector. The59-end primer, 59-GGATCCCCCGGG(C/T)AGAAAAAGCAAACCG, wasdegenerate at the designated position. It overlapped the XmaI site(underlined) and extended 7 nt upstream into the parent vector. AllpML16 series templates were sequenced for verification.Elongation Factors—Human recombinant SII (rSII) purified as de-

scribed previously (18) was either generously furnished by RobertLandick (Washington University, St. Louis) or else made within ourlaboratory using the pET11d-RAP38 expression vector kindly suppliedby Zachary Burton (Michigan State University, East Lansing).Assembly and Purification of Stalled and Arrested Elongation Com-

plexes—The complete procedure has already been described (11, 15). Toform preinitiation complexes, DNA templates were incubated in HeLacell nuclear extract, after which a gel filtration step was performed topartially purify the complexes and separate out residual NTPs. Formost experiments, the initial transcription was done in the absence ofGTP, since the nontemplate strand in all of our plasmids lacks Gresidues over at least the initial 15 bases. The initiating and labelingnucleotides are given in each figure legend. For CTP labeling, transcrip-tion reactions contained either 100 mM ATP or 1–2 mM ApC (dinucle-otide-primed reactions also contained 10 mM dATP), along with 10 mM

UTP and 0.5 or 1 mM [a-32P]CTP; after 5 min at 25 °C, unlabeled CTPwas added to give a final CTP concentration of 10 mM and incubationwas continued for another 5 min. For UTP labeling either 120 mM ATPor 2 mM CpA plus 10 mM dATP was added along with 10 mM CTP and 0.5or 1 mM [a-32P]UTP; after 5 min at 25 °C, unlabeled UTP was added to10 mM and incubation was continued for another 5 min. Complexesstalled at 136 on template pML5-MUT3 were generated using 2 mM

CpA, 10 mM dATP, 1.0 mM [a-32P]UTP and 10 mM CTP and GTP, followedby 10 mM UTP chase. The initial, labeled complexes were purified by aprocedure we have called Sarkosyl rinsing (see Izban and Luse (15) fora complete description). This involves the addition of 1% Sarkosylfollowed by gel filtration on Bio-Gel A1.5m in 30 mM Tris-HCl, pH 7.9,10 mM b-glycerophosphate, 62.5 mM KCl, 0.5 mM EDTA, and 1 mM

dithiothreitol. Sarkosyl-rinsed complexes lack free transcription factorsand free NTPs.Complexes stalled at 1155 on templates pML20-U158 and pML20-

U160 were produced exactly as described elsewhere (16). Complexesarrested at 1194 on pML5–4NR were assembled somewhat differentlyin each of the three experiments where they were used. The arrestedcomplexes shown in Figs. 3 and 7A were generated as described previ-ously (16) except as noted in the figure legends. To generate uniformlylabeled complexes for Fig. 7B, stalled complexes C15 and U18 wereassembled as described previously (11) except that initiation was per-formed with 0.25 mM each of a-32P-labeled and nonlabeled CTP and 120mM ATP in place of ApC and dATP. Uniformly labeled arrested com-plexes were then produced exactly as described previously (16).Chase Reactions—Elongations in excess NTPs were all performed at

37 °C for the times specified in the figures. The reactions includedMgCl2 at 8 mM and the four nucleoside triphosphates at 1 mM unlessotherwise noted. When a time course was run, all aliquots were with-drawn from a common pool. Chase reactions in the presence of elonga-tion factor SII were set up with that factor at 1.5 mg/ml. Chases in thepresence of a-amanitin (at 1 mg/ml) were preceded by incubating thecomplexes with a-amanitin at 37 °C for at least 3 min before addingother reagents. RNAs were purified as described elsewhere (15) exceptthat Proteinase K treatment was for 0.6–1.25 h at room temperatureand RNAs were boiled for 2.5 min before loading. Samples were re-solved on denaturing polyacrylamide gels consisting of 7% (19:1), 10%(29:1), 15% (29:1), 20% (19:1), or 28% (25:3) acrylamide/bisacrylamideand visualized either by autoradiography or by use of a PhosphorIm-ager (Molecular Dynamics) as described previously (15, 16). Exactlength markers for Fig. 1 were generated with sets of NTP-limitingtranscription reactions (not shown) synthesized on the same template(see Izban et al. (19)). The reader will note, in Fig. 1A, that RNAssynthesized on pML16220 do not comigrate with RNAs made onpML16C27 or pML16T27. This difference was completely reproducibleand presumably results from the single base change at 127 on thenontemplate strand. All three of these templates were carefully se-quenced to confirm that no changes occurred in creating pML16C/T27from pML16220 except at position 127.Transcript Cleavage Reactions—Cleavage reactions with SII were

done essentially as described previously (11, 16), using rSII at theconcentrations and for the incubation times indicated in the figurelegends. Pyrophosphate-facilitated cleavage reactions, in the presenceor absence of a-amanitin, were all performed with 2 mM sodium pyro-phosphate at 37 °C for the times indicated. Incubations with Mg21

alone contained 8 mM MgCl2 and continued at 37 °C for the timesindicated.

RESULTS

Experimental System—We study transcript elongation byRNA polymerase II in vitro with partially purified ternarytranscription complexes. The initial transcribed regions of ourtemplates are designed so that in the presence of a subset of theNTPs, newly initiated RNA polymerases will pause between 15and 25 nt downstream of transcription start. These complexesare sufficiently stable to allow purification by transient expo-sure to the detergent Sarkosyl during gel filtration, a procedurewe call Sarkosyl rinsing (15). The large majority of the Sarko-syl-rinsed complexes will resume transcription when NTPs areadded. These complexes lack the TFIIF and SII elongationfactors. However, when these factors and NTPs are added insaturating amounts, the purified complexes will elongate theirnascent RNAs at about 1500 nt/min at 37 °C (20), which isessentially the chain elongation rate observed in the cell nu-cleus (21). We use the term “stalled” for complexes which havestopped transcription because of the absence of NTPs butwhich remain competent to resume RNA synthesis rapidlywhen NTPs are restored. Stalled complexes are named accord-ing to the length of the nascent RNA and the last base incor-porated; thus, a complex with a 23-nt RNA ending in C wouldbe a C23 complex.

a-Amanitin Slows but Does Not Absolutely Block TranscriptElongation—In the course of recent experiments we observedan example of amanitin-resistant chain elongation by RNApolymerase II which was much more extensive than thosereported previously (8–10). The reaction which sparked ourinterest was performed on the pML16220 template, which hasno T residues on the nontemplate strand from 121 through1155 (see Fig. 1A). Transcriptions performed in the absence ofGTP gave complexes paused at 123 (C23 complexes). Thesecomplexes were Sarkosyl-rinsed and a portion were treatedwith amanitin at 1 mg/ml for 3 min at 37 °C. (This preincuba-tion protocol was used for all amanitin-containing reactions inthis study.) We found that amanitin-treated C23 complexesmade an average of 11 or 12 bonds in 5 min at 37 °C whenincubated with all four NTPs at 1 mM; some complexes synthe-sized as many as 30 bonds in 20 min under these conditions(Fig. 1A, lanes 1–5). Most of the RNAs made by noninhibitedcontrol complexes were too long to resolve on the gel shown inFig. 1A. RNA synthesis in the presence of the toxin can con-tinue for at least 2 h at 37° (Fig. 1B, lane 6).The pattern of products obtained on the pML16220 template

in the presence of a-amanitin was reproducible in many exper-iments using different batches of amanitin and nuclear extract.Amanitin was reported to have a very slow off-rate from RNApolymerase II at 37 °C (1.2 3 1024/s; see Cochet-Meilhac andChambon (4)), but those experiments were done under differentconditions from those we employed. We were concerned thatthe catalytic activity of the polymerase in the presence of thetoxin at 37 °C might reflect cycling of the drug between solutionand polymerase, rather than low activity of the polymerasewhen amanitin is bound. To address this, we repeated theexperiment shown in lanes 1–5 of Fig. 1A, except that after theaddition of a-amanitin another round of gel filtration was per-formed on the C23 complexes to remove free amanitin. Wefound that in a 20-min chase the majority of these C23 com-plexes behaved identically to those in lane 5 of Fig. 1A; how-ever, about one-third of the complexes transcribed much more

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FIG. 1. RNA polymerase II complexes still elongate in the presence of a-amanitin but at a reduced rate. A, C23 complexes on thepML16220 template (lanes 1–5) or on pML16220 variants having a T (pML16T27; lanes 6–10) or a C (pML16C27; lanes 11–15) at position 127 onthe nontemplate strand were pretreated with a-amanitin as indicated and chased with 1 mM NTPs for the times specified. The initial transcriptionreaction contained 2 mM ApC and 0.5 mM [a-32P]CTP. B, C23 complexes on the pML16220 template were amanitin-treated and chased with 1 mM

NTPs as indicated. The initial transcription reaction contained 100 mM ATP and 1 mM [a-32P]UTP. C, C23 complexes prepared as in B were chasedwith or without amanitin using the times and NTP concentrations indicated. For all panels, the RNA products were purified and resolved on 20%polyacrylamide gels as described under “Materials andMethods.” Pertinent transcript lengths generated on pML16220 are presented together withtemplate sequence on the left of panel A and on the right of panel B; lengths of various transcripts produced on pML16T27 and pML16C27 arepresented together with the template sequence on the right of panel A.

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rapidly, at the same rate as uninhibited controls (data notshown). This is consistent with the rate of release of amanitinfrom polymerase II measured in earlier studies (4). These re-sults indicate that the ability of RNA polymerase to make 10 ormore bonds in a 5-min incubation in the presence of amanitincannot be explained by rapid binding and release of the toxin.The results in Fig. 1 showed that chain elongation proceeded

efficiently through the initial 12 bases downstream of 123 butslowed as the polymerase passed through 136 to 143 (Fig. 1A,lanes 3–5), and again when it passed through 154 to 161 (Fig.1B, lanes 3–6). This reduction in rate appeared to correlatewith the requirement for incorporation of C residues into thegrowing RNA. To begin to address this point more directly, weperformed a number of experiments. First, we created twovariants of pML16220 in which the A on the nontemplatestrand at 127 was replaced with a T or a C residue. Transcriptelongation was delayed after incorporating a C at 127 (Fig. 1A,lane 13), while elongation was delayed both before and afterincorporating a U at this position (Fig. 1A, lane 8). This sug-gested that amanitin is differentially affecting the ability ofpyrimidines and purines to be incorporated into RNA. How-ever, this result could also mean that RNA polymerase IInormally incorporates pyrimidines more slowly, but such be-havior is not easily detected because of the rapid rate of tran-script elongation in the absence of amanitin. To test this idea,we again chased C23 complexes on the pML16220 template inthe presence and absence of amanitin. In this case, the rate oftranscript elongation in the reactions lacking amanitin wasconsiderably slowed by reducing the concentration of the chaseNTPs to 25, 50 or 100 mM. The results (Fig. 1C) show that mostof the pauses which occur in the presence of amanitin over a 2-htime course are also observed over the first 10 s to 1 min inreactions performed at suboptimal NTP levels without aman-itin (compare lanes 5 and 12, and lanes 6, 11, and 13). Not allpause sites were seen under both conditions, and the relativelevel of pausing did vary at some sites, for example at position160 (compare lanes 5 and 9).The ability of RNA polymerase II to cross long stretches of T

residues on the nontemplate strand was also investigated,which required the use of a different template. We constructeda plasmid in which the sequence of the nontemplate strandfrom 144 downstream reads: 59. . .TTTTTTCTCCATTT-TA. . .39. U36 complexes assembled on this template were Sar-kosyl-rinsed and then chased with NTPs at 37 °C. Althoughmost complexes in a toxin-free control reaction cleared bothT-runs within 5 min (Fig. 2, lane 3), essentially all amanitin-treated complexes remained near the end of the first T-runeven after 2 h (Fig. 2, lane 9). Thus, amanitin appears to slowtranscription very effectively during the synthesis of U-richsegments of the transcript, producing an effect similar to tran-scriptional arrest. This result was not unexpected, since we hadpreviously shown (16) that stalling RNA polymerase transcrip-tion complexes after the addition of more than 3 consecutive Uresidues to the nascent RNA can lead to arrest. This point willbe explored further under “Discussion.”Amanitin could slow chain elongation either by affecting

bond formation itself or by retarding the ability of the activesite to translocate along the template. If translocation were theprimary target of amanitin, and if complexes stalled by NTPstarvation were already poised to add the next base (that is, iftranslocation of the active site had already taken place), thenaddition of the initial base in the presence of amanitin wouldproceed at the normal rate. We tested this idea with C23complexes produced on the template used in Fig. 1B. While themajority of uninhibited control complexes left the starting po-sition and added several nucleotides within 15 s, amanitin-

treated complexes made no bonds in 15 s. The majority of thetreated complexes had made no bonds even after 60 s (data notshown). This is consistent with the idea that amanitin blocksbond formation, but it is also possible that stalled complexesmust first translocate the active site in order to add the nextNTP, in which case translocation could be the step affected byamanitin.

a-Amanitin Exacerbates the Arrested Condition—Many lab-oratories have observed (11, 22–25) that transcription throughcertain DNA sequences causes a fraction of the RNA polymer-ase II ternary complexes to pause without termination. Thesearrested complexes resume RNA synthesis very slowly (i.e.from 10s of minutes to hours) in the presence of NTPs alone;however, the transcriptional competence of arrested complexesis rapidly recovered by treatment with elongation factor SII(11, 22, 24, 25). We have shown that on the pML5–4NR tem-plate arrest occurs 194 bases downstream of the transcriptionstart, within a stretch of T residues on the nontemplate strand(16). This observation is reproduced in Fig. 3. Complexes werearrested at 1194, and NTPs were removed by gel filtration(lane 1). These U194 complexes resumed elongation rapidly inthe presence of SII and NTPs (lane 5; see also Izban and Luse(16)). In the absence of SII, a much longer time was needed toclear the arrest site (lanes 6–8). Note that the chases in lanes6–8 were performed with ATP, CTP, and UTP only; we pre-sume that the slow leakage through G-stops at 1207 andbeyond resulted from GTP contamination in the other nucleo-tides. When the arrested complexes were challenged withNTPs after amanitin treatment, essentially no resumption oftranscription from position 1194 was seen, even after 2 h (lane11). Thus, in contrast to complexes stalled by NTP starvation,arrested complexes are inactivated for further chain elongationby a-amanitin, at least over a time course of several hours.Again, this result was anticipated; since the rate of escape fromarrest by resumption of bond formation is normally very slowand bond formation rates are drastically reduced with aman-itin, there should be essentially no detectable escape fromarrest in the presence of the toxin over the reaction times weemployed.While the arrested complexes cannot continue RNA synthe-

FIG. 2. a-Amanitin causes RNA polymerase II to pause se-verely when transcribing through a T-run. All reactions used U36complexes (lane 1) assembled on the pML5-MUT3 template. The initialtranscription reaction contained 2 mM CpA and 1 mM [a-32P]UTP. Onealiquot was incubated with 8 mM MgCl2 for 120 min (lane 2) and asecond was incubated with 1.5 mg/ml SII and 1 mM NTPs, for 7 min(lane 10). The remaining complexes, with or without a-amanitin asnoted, were chased with 1 mMNTPs for the indicated times. Transcriptswere resolved on a 15% polyacrylamide gel. Relevant transcript lengthsand the associated template sequence are displayed in the right margin.

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sis in the presence of amanitin, the results in Fig. 3 stronglysuggest that transcript cleavage in these complexes is not sen-sitive to the toxin (compare lanes 3 and 4, or lanes 7 and 8, or10 and 11). We will consider the question of amanitin’s effect ontranscript cleavage in more detail in a later section.

a-Amanitin Increases the Likelihood That Complexes Proneto Arrest Will Fall into That Condition—Our laboratory previ-ously showed that complexes stalled after adding a poly(U)segment (U tail) to the end of the nascent RNA behave progres-sively more like arrested complexes as the U tail is lengthened(16). Complexes stalled with a 39 end consisting of 3 U residuesresumed elongation after a 2-min incubation with excess NTPs.However, 40% of complexes with an otherwise identical nas-cent RNA having 5 U residues at the 39 end did not resumeRNA synthesis when chased for 2 min (16). We used the sametemplates employed in the earlier study to investigate theeffect of a-amanitin as a function of the length of the U tail.Sarkosyl-rinsed complexes were chased to the end of a U-freecassette at 1155 on templates in which either the next 3(pML20-U158) or the next 5 (pML20-U160) residues on thenontemplate strand are Ts (see Fig. 4). The G155 complexeswere then gel-filtered and challenged with various combina-tions of NTPs, with or without a-amanitin. Most complexes onboth templates resumed elongation from 1155 when chasedwith all NTPs (Fig. 4, lanes 3 and 13), or when chased to theG-stop at 1159 (lane 4) or 1160 (lane 8). The large majority ofC159 complexes on the U158 template resumed transcriptionafter adding G and incubating for 5 min at 37 °C (lane 6), butless than half of those starting from 1160 on the U160 tem-plate did so (lane 10), as expected from previous studies (16). It

is important to note that the sequence immediately down-stream of 1159 on the U158 template and 1160 on the U160template is the identical DNA segment, containing only pu-rines on the nontemplate strand, which is present downstreamof position 123 on the template used in Fig. 1. Thus, one wouldexpect that C159 complexes on the U158 template and U160complexes on the U160 template should chase effectively in 5min in the presence of a-amanitin. However, most of the C159complexes and nearly all of the U160 complexes failed to re-sume elongation after amanitin treatment (compare lanes 4and 5, and lanes 8 and 9). If the complexes were advancedfurther along the template before amanitin addition, such thatthe 39 ends of the nascent RNAs were no longer U-rich (com-plexes G163 on the U158 template and G164 on the U160template), elongation in the presence of amanitin was onceagain efficient (lanes 6 and 7, and 10 and 11). These resultssuggest that the effect of amanitin depends not only on thesequence of bases to be added to the RNA but also on thesequence at the 39 end of the nascent transcript. It is worthnoting that in the presence of a-amanitin RNA polymerase IIsynthesized the polypurine segment of RNA between, for ex-ample, 1160 and 1172 on the U158 template about as rapidlyas it synthesized the nearly identical RNA (between 124 and135) on the template in Fig. 1. Thus, the ability of transcrip-tion to proceed at a greatly reduced rate in the presence ofamanitin is not strongly affected by the distance downstream oftranscription start.The Sequence Composition at the Nascent Transcript’s 39 End

Is a Crucial Determinant of a-Amanitin Inhibition—To furtherexplore the role of the transcript in amanitin inhibition, weperformed an experiment in which we could compare the aman-itin response of transcription complexes that differed only inthe 39 ends of their nascent transcripts. The first G residuesdownstream of transcription start on the nontemplate strand ofthe pML16220 template (see Fig. 1) occur at positions 124through 126. Thus, incubation of C23 complexes with eitherGTP or ITP should generate G26 or I26 complexes. The differ-ent mobilities of the G26 and I26 transcripts confirmed thatIMP was successfully incorporated in place of GMP (Fig. 5,compare lanes 5 and 6). As expected from the results shown inFig. 1, the G26 complexes showed substantial chain elongationin 5 min in the presence of a-amanitin (lane 11). However, theI26 complexes, most of which chased in the absence of amanitin(lane 8), were inactive in a 5-min elongation reaction in thepresence of the toxin (lane 9). This was not the result of a blockof ITP incorporation by amanitin, since the first two basesadded to the 126 complexes are A residues (see Fig. 1A). Notealso that the initial chase from 123 to 126 could be completedwith ITP in the presence of amanitin (lane 3). Thus, transcrip-tion complexes which are identical except for the three residuesat the 39 end of the nascent RNA can have very differentresponses to a-amanitin.

a-Amanitin Greatly Reduces the Rate of Pyrophosphorolysisin Stalled Complexes—Stalled RNA polymerase II transcrip-tion complexes incubated with pyrophosphate liberate NTPs bysequential cleavage of NMPs from the 39 ends of the nascentRNAs (13, 26). Stalled and arrested ternary complexes can alsocleave their nascent transcripts without the addition of pyro-phosphate or other factors (11, 12, 27). We believe this repre-sents an intrinsic activity of the RNA polymerase and notresidual contamination with SII; this point will be consideredin detail under “Discussion.” Complexes stalled at 120 on thepML20 template were incubated for 2, 15, or 60 min either withMg21 alone or with Mg21 and 2 mM pyrophosphate; for eachcondition reactions were performed with or without a-amanitin(Fig. 6). After 2 min, substantial pyrophosphorolysis was ob-

FIG. 3. The effect of a-amanitin upon arrested complexes.U194complexes (lane 1) were prepared on the pML5–4NR template; initialtranscription reactions contained 1 mM ApC and 1 mM [a-32P]CTP.Complexes were mixed with RNAsin (0.4 unit/ml), pretreated witha-amanitin (lanes 4, 9, 10, and 11) and then incubated for the specifiedtimes. In lane 2, no additions were made; all other lanes received 8 mM

MgCl2. NTPs were added to 1 mM and SII to 1.5 mg/ml where indicated.Transcripts were electrophoresed on a 10% polyacrylamide gel. Signif-icant transcript lengths are noted in the right margin; values from 207to 236 denote the location of G stops downstream of 1194.

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served, compared with the control, which received only Mg21

(lanes 2 and 3), and this reaction was nearly completely aman-itin-sensitive (compare lanes 4 and 5). After 15 min, however, itwas clear that some pyrophosphorolytic cleavage did take placeabove the Mg21-only background in the presence of amanitin.Quantitation of the remaining 20-mer indicated that only 46%as much uncleaved 20-base transcript remained in lane 9 com-pared to lane 8. Thus, as we observed with the forward reac-tion, pyrophosphorolysis in stalled complexes was strongly butnot completely inhibited by amanitin. Cleavage in the Mg21-only case was also reduced by amanitin. After 2 min, cleavagewas completely blocked (compare lanes 3 and 4); after 15 min,some cleavage had taken place in the presence of amanitin(lane 8) but the amount of uncleaved transcript in lane 7 (noamanitin) was only 51% of the amount in lane 8, indicating thattranscript cleavage in the Mg21-only reaction was not com-pletely amanitin-sensitive. Note in lanes 11 and 12, wherecleavage with Mg21 had continued for 1 h, that amanitin re-duced the total amount of cleavage (the ratio of 20-mer in lanes11 and 12was 0.44), and it also strongly reduced the productionof the 19-mer (compare also lanes 7 and 8 from the 15-minreaction). However, the production of cleavage products shorterthan 19 was actually greater in the presence of amanitin after1 h, even though total cleavage, as judged by the amount of20-mer remaining, was reduced. We are not certain of thereason for this. It suggests that amanitin can inhibit only theinitial spontaneous cleavage in a stalled complex; once this cutis made, subsequent cleavages are resistant. It is possible thatthe 19-mer is a metastable species. If cleavage in the presenceof amanitin must bypass the 19-mer, subsequent cleavageevents may be easier.Finally, SII-mediated transcript cleavage in amanitin-

treated stalled elongation complexes was completely blocked bythe toxin in all cases (data not shown), in agreement withearlier results (11).

a-Amanitin Inhibits Neither the Intrinsic Cleavage Activitynor Pyrophosphate-mediated Transcript Cleavage by ArrestedTernary Complexes—We have shown that complexes arrestedat 1194 on the pML5–4NR template cleave 7–17 nt from the 39ends of their nascent RNAs in the presence of SII (13, 16). Thesame set of 7–17 nt RNAs are released at a much slower rate

when the U194 complexes are incubated with Mg21 alone (13).Pyrophosphate treatment of U194 complexes also results in therelatively rapid release of the 7–17-nt RNAs; in this case theliberated fragments have 59-triphosphate termini (13). As ex-pected from previous reports (11, 12), SII-mediated transcriptcleavage in U194 complexes was completely blocked bya-amanitin (data not shown). The results in Fig. 3, however,indicated that amanitin had no effect on the intrinsic transcriptcleavage activity of arrested complexes. A 2-h incubation of1194 complexes with Mg21 gave the same level of truncationproducts in the presence or absence of amanitin (Fig. 3, lanes 3and 4). The major shortened transcripts in these lanes ap-peared to correspond to the major 10- and 14-base cleavagesobtained within minutes in SII-mediated truncation reactionsof the 194-nt transcript (data not shown for this figure; see Fig.7B and Rudd et al. (13) and Izban and Luse (16)). As expected(11, 12), transcript cleavage in arrested complexes led to reac-quisition of elongation competence, so that intrinsic cleavage inthe presence of amanitin, ATP, CTP, and UTP resulted inelongation up to the first G-stop upstream of the arrest site, atposition 1186 (Fig. 3, lanes 10 and 11).To confirm that amanitin has no effect on the endogenous

cleavage reaction, we decided to examine directly the frag-ments released from the 39 ends of the nascent RNAs. Weprepared U194 complexes whose nascent RNAs were uniformlylabeled with [32P]UTP and incubated them for 60 min in Mg21,with or without a-amanitin. RNAs liberated in this reactionwere resolved on the gel shown in Fig. 7B. For reference, lane7 contains RNAs produced by SII-mediated transcript cleavage.As expected (13), lower levels of these same RNAs were ob-tained in Mg21-only incubations (lane 3). U194 complexestreated with amanitin gave the same level of cleavage productsas the noninhibited complexes (compare lanes 3 and 4). Thus,factor-independent transcript cleavage in arrested complexes isamanitin-resistant.Pyrophosphorolysis in arrested U194 complexes occurred in

the presence of a-amanitin (Fig. 7A, compare lanes 2 and 3).However, while the major cleavage at 14 nt from the 39 endappeared to occur to about the same extent with or without thetoxin, other aspects of the cleavage pattern differed betweenthe reactions. Since the initial cleavage produces an elonga-

FIG. 4. Sensitivity to a-amanitin increases when the transcript ends with many U residues. G155 complexes were prepared on eitherthe pML20-U158 (lane 1) or pML20-U160 (lane 15) templates; the initial transcription reactions contained 2 mM ApC and 0.5 mM [a-32P]CTP.Complexes were either incubated with 8 mM MgCl2 alone for 14 or 16 min (lanes 2 and 14, respectively), chased with 100 mM each of all NTPs for5 min (lanes 3 and 13, respectively) or walked forward in 3-min incubations, first with 100 mM UTP (lanes 8–11) or with 100 mM each of UTP andCTP (lanes 4–7), next with 100 mM GTP (lanes 6, 7, 10, and 11), and finally with 100 mM ATP (lane 12). Aliquots of complexes stalled at the positionsindicated were preincubated with amanitin and then chased with 100 mM each of all NTPs for 5 min (lanes 5, 7, 9, and 11). RNAs were resolvedon a 7% polyacrylamide gel. Part of the RNA sequence encoded by each of the two templates is shown along the lower margin, and pertinenttranscript lengths are displayed in the left margin.

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tion-competent complex which performs subsequent pyrophos-phorolysis very slowly (Fig. 6), the difference might reflectmultiple pyrophosphorolytic cleavages in the absence of thetoxin versus a single cleavage in its presence. We resolved thispoint by examining the short RNA fragments released by in-cubation of uniformly labeled U194 complexes with pyrophos-phate. These RNAs were found to be nearly identical (exceptingonly a single RNA of about 5 nt) regardless of the presence ofamanitin in the incubation (Fig. 7B, compare lanes 5 and 6).Thus, endonucleolytic transcript cleavage by arrested com-plexes in the presence of pyrophosphate is not sensitive toa-amanitin.

DISCUSSION

We have found that the mushroom toxin a-amanitin sub-stantially reduces the rate of transcription by elongation com-petent RNA polymerase II ternary complexes; however, elon-gation was not completely blocked. Most amanitin-treatedcomplexes can continue elongation for hours, but complexeswhich are arrested are essentially unable to resume transcrip-tion in the presence of amanitin. While amanitin greatly re-tards pyrophosphorolysis by elongation-competent complexes,

it has no effect on either the intrinsic or pyrophosphate-medi-ated endonucleolytic transcript cleavage activities of arrestedcomplexes. After this study was submitted, Chafin et al. (28)reported that Drosophila RNA polymerase II initiated frompoly(dC)-tailed templates also elongates RNA chains and per-forms pyrophosphorolysis at greatly reduced but detectablerates in the presence of a-amanitin.It had been observed previously that promoter-initiated RNA

polymerase II elongation complexes treated with amanitin canadd several nucleotides to their nascent chains (8, 9). Recently,Gu et al. (10) reported that a specifically initiated RNA polym-erase II ternary complex stalled at 1218 or 1220 could con-tinue transcription for about 8 bases in the presence of aman-itin. An examination of the sequence of the RNA-like strand ofthe template used by Gu et al. downstream of 1218/220 showsthat all but one of the next 9 or 11 bases are purines, followedby three pyrimidines (10). Thus, it seems likely that Gu et al.observed the same effect which we document here. We specu-late that the relative rarity of sufficiently long purine runs inrandom sequence DNA accounts for the lack of previous reportson the incomplete inhibition of transcript elongation by aman-itin (but see Job et al. (29), discussed below).We have reported that RNA polymerase II initiating at the

adenovirus 2 major late promoter cannot add even a singlenucleotide to a dinucleotide primer in the presence of amanitin(30). In those experiments, production of a low level of trinu-cleotide was observed with amanitin, but this same level wasalso seen even in the absence of template, or with dinucleotidesthat could not prime RNA synthesis at the adenovirus pro-moter. We interpreted these results to mean that the amanitin-resistant trimer was generated by activities other than RNApolymerase II. However, in light of our current results wecannot exclude the possibility that a very low level of transcrip-tion initiation can take place in the presence of amanitin. RNApolymerase II preinitiation complexes assembled from nuclearextracts are unstable in the presence of ATP, which is requiredfor transcription initiation under these conditions (30, 31).

FIG. 5. Response to a-amanitin is influenced by the sequenceat the nascent transcript’s 3* end. C23 complexes were assembled onthe pML16220 template (lane 1); the initial transcription reaction con-tained 120 mM ATP and 0.5 mM [a-32P]UTP. The C23 complexes wereincubated 1 min with 1.6 mg/ml SII and 8 mM MgCl2 (lane 2) or chasedto position 126 with either 20 mM GTP for 3 min (G26 complexes, lane5) or 1 mM ITP for 15 min (I26 complexes, lane 6). Other C23 complexeswere pretreated with a-amanitin and chased for 15 min with 1 mM ITP(lane 3). A portion of the I26 complexes was supplied with 1 mM each ofATP, CTP, and UTP for an additional 5 min incubation (lane 4). OtherI26 complexes were incubated 1 min with 1.6 mg/ml SII and 8 mMMgCl2(lane 7); the rest were chased 5 min with 1 mM standard NTPs with orwithout a-amanitin, as indicated (lanes 8 and 9). The G26 complexeswere identically chased with or without a-amanitin, as indicated (lanes10 and 11). Transcripts were resolved on a 20% polyacrylamide gel.Pertinent RNA lengths are provided in the right margin, with 26(I) and26(G) denoting 26-nt products containing I or G residues, respectively,at their 39 end.

FIG. 6. a-Amanitin slows pyrophosphorolysis of stalled com-plexes. U20 complexes were assembled on the pML20 template (lane1); the initial transcription reaction contained 2 mM ApC and 1 mM

[a-32P]CTP. The complexes were incubated with 8 mM MgCl2 alone(lanes 3, 4, 7, 8, 11, and 12), or with 8 mM MgCl2 and 2 mM PPi (lanes2, 5, 6, 9, 10, and 13), with or without a-amanitin for the times indi-cated. The purified products were resolved on a 28% polyacrylamide gel.The position of the 20-nt transcript is indicated in the right margin.

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Thus, amanitin-treated preinitiation complexes might be inac-tivated before any bonds could be formed.The effects of amanitin on transcription of homopolymeric

templates by pure RNA polymerase II have also been studied.Two groups reported that a-amanitin permits formation of thefirst phosphodiester bond but absolutely blocks the synthesis ofall subsequent bonds (32, 33). It is difficult to compare theseresults to our own findings since the RNA polymerases in thesesystems did not pass through initiation at a promoter. Onestudy on transcription of homopolymeric templates detectedextensive amanitin-resistant transcription by polymerase II.Job et al. (29) reported that with poly(dC) or poly(dC)zpoly(dG)as template and GTP as substrate, about 30% of RNA synthesisby wheat germ RNA polymerase II was resistant even to 100mg/ml a-amanitin. Slippage of the nascent transcript on thetemplate clearly played a part in these results (29), but it isinteresting that the only template-substrate combinationwhich allowed significant amanitin-resistant transcription in-volved the synthesis of a polypurine transcript.We found that the sequence of the region to be transcribed

was a major factor in the ability of RNA polymerase II to extendthe nascent RNA, in the presence or absence of amanitin (Fig.1, A-C). However, the results of substituting ITP for GTP inonly the last three bases of the nascent RNA (Fig. 5) argue thatthe sequence of the transcript is also important in elongationcompetence. In order to rationalize these observations it isuseful to briefly review current ideas on the mechanism oftranscriptional arrest. Several groups have noted that arrestby RNA polymerase II most often occurs immediately after thesynthesis of a U-rich RNA (20, 22, 23, 34–38). Sequences flank-ing the template region encoding the poly(U) segment also playa very important role in arrest (23, 34, 39, 40). We had notedthat DNA with 5 consecutive T residues on the nontemplatestrand does not provide a barrier to the polymerase duringtranscription with excess NTPs. However, if the polymerasewas forced to pause after the incorporation of the 5 U residues,because of the absence of the next NTP required for elongation,then nearly half of the complexes could not resume transcrip-tion after a 5 min incubation with excess NTPs (16). Thus,while the incorporation of many consecutive U residues doesnot necessarily force arrest, polymerases crossing T-rich sec-tions of the nontemplate strand are in danger of arrest. Ourresults suggested that the length of time which the polymerasespends with a transcript containing a U-rich 39 end is crucial tothe arrest process. Very recently, the importance of “dwelltime” at potential arrest sites was directly assessed by chang-ing the overall rate of transcription with TFIIF or ammoniumions; in both cases, more rapid transcription was inverselycorrelated with arrest (41).Once arrest has occurred, rapid resumption of transcription

cannot take place without cleavage of the nascent RNA wellupstream (from 5 or 6 to as many as 17 bases) of the initial siteof bond formation (16, 42). Although transcript cleavage occursspontaneously in both stalled and arrested complexes, it isgreatly stimulated by the SII elongation factor (11–13). Thesource of this spontaneous cleavage is still somewhat contro-versial. We have argued that this activity is intrinsic to theRNA polymerase itself. While we cannot absolutely eliminatethe possibility of SII contamination in our partially purifiedcomplexes, the following points argue strongly against it. First,if the cleavage were caused by a very low level of residual SII,which was not removed by gel filtration in the presence ofSarkosyl, one would expect that a second round of gel filtrationunder the same conditions would remove almost all of theresidual activity. However, when we did such an experiment,we did not see any reduction in cleavage levels after the com-

FIG. 7. Both intrinsic and pyrophosphate-mediated transcriptcleavage are a-amanitin-resistant in arrested complexes. A, ar-rested U194 complexes on the pML5–4NR template (lane 1) were eitherincubated for 5 min with 1.5 mg/ml SII and 1 mM NTPs (lane 4) orincubated for 16 min with 8 mM MgCl2 and 2 mM pyrophosphate (lanes2 and 3), with or without amanitin as specified. Transcripts wereelectrophoresed on a 10% polyacrylamide gel. The initial transcriptionreaction contained 100 mM ATP and 1 mM [a-32P]CTP. B, arrested U194complexes on the pML5–4NR template (lane 1) were generated withuniformly labeled transcripts as described under “Materials and Meth-ods.” The initial transcription to generate C15/U18 complex contained120 mM ATP and 0.25 mM each of [a-32P]CTP and nonlabeled CTP; theinitial chase reaction contained 20 mM [a-32P]UTP. The U194 complexeswere either pretreated with a-amanitin or not, as noted, and thenincubated for the specified times, either without additional reagents(lane 2), with 8 mM MgCl2 alone (lanes 3 and 4), with 8 mM MgCl2 and2 mM pyrophosphate (lanes 5 and 6), or with 8 mM MgCl2 and 1.5 mg/mlSII (lane 7). The cleavage products were purified and electrophoresedon a 22.5-cm long 28% polyacrylamide gel. The lengths of relevanttranscripts (panel A) or SII-mediated cleavage products (panel B) arenoted in the right margins of the respective panels.

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plexes had been subjected to a second gel filtration step (datanot shown). Second, the factor-independent cleavage activity inFig. 6 makes its initial cut only one nucleotide from the 39 end;however, the addition of SII to stalled complexes leads to cleav-age primarily in dinucleotide increments (26). The action ofamanitin on arrested complexes also argues against contami-nation. We can see no stimulation of cleavage by added SII inthe presence of amanitin, and yet the spontaneous cleavageactivity is completely amanitin-resistant. This might be ex-plained if amanitin could only inhibit the binding of SII; in thismodel, residual SII, which is already bound, would not beinhibited. However, when we tested this idea by adding SII tocomplexes and then followed with amanitin, we still saw abso-lutely no cleavage above the control (data not shown). Finally,it is important to recall that Escherichia coli RNA polymeraseshows spontaneous transcript cleavage activity even when it isprepared from cells which lack functional genes for both theGreA and GreB transcript cleavage factors (43). RNA polymer-ase III, which has no known elongation factors, also exhibitsspontaneous cleavage of transcripts in stalled complexes in thepresence of Mg21 (44).We had hypothesized that arrest might result from loss of

contact between the active site of the polymerase and the 39 endof the transcript (16). Transcript cleavage was seen as a mech-anism to generate a new 39 end that is accessible to the activesite. The subsequent demonstration that pyrophosphate canalso stimulate cleavage at the same sites as SII suggested thatthe active site itself might be the cleavage agent (13). Arrestcould then reflect the translocation of the RNA polymerase’scatalytic center upstream along the nascent RNA. It is plausi-ble that U-rich regions are the least avidly bound by the activesite, making transcription complexes with U-rich 39 ends themost prone to arrest. Elongation competence would be restoredby SII-stimulated cleavage at upstream locations on the tran-script with which the active site stably associates.In the context of this model (see also Gu and Reines (41)) the

importance of dwell time at potential arrest sites is easy toenvision. If upstream translocation of the active site is muchslower than the usual rate of bond formation, arrest will bevery unlikely unless the polymerase can be paused for sometime after synthesis of the crucial U-rich 39 end. Thus, in Fig.4, a C159 complex whose transcript ends . . .GUUUC-39 ismostly active when chased, but when amanitin is added togreatly increase dwell time (by lowering the rate of initial bondformation), most of the C159 complexes are inactive upon chase(compare lanes 4–6). A U160 complex, with a more U-rich 39end (. . .GUUUUU-39), is only partially active in the absence ofamanitin and nearly inactive in the presence of the toxin (com-pare lanes 8–10 of Fig. 4). We had suggested (16) that theactive site probably partitions between elongation-competentand elongation-incompetent locations in arrested complexes,since arrested complexes show a very slow but easily detectedrate of resumption of transcription (Fig. 3, lanes 6–8). Bondformation at 37 °C and 1 mM NTPs occurs on average about 5times/s (20), so the active site would need to be in the elonga-tion competent configuration for only a very short period toallow some complexes to escape from arrest. However, bondformation is much slower in the presence of amanitin, whichwould account for the inability of amanitin-treated, arrestedcomplexes to resume elongation (Fig. 3).What conclusions can we draw concerning the mechanism of

inhibition of transcription by amanitin, given both our presentresults and the large body of earlier work on the toxin? Previ-ous studies with homopolymeric templates (29, 32, 33) hadsuggested that translocation and not bond formation is blockedby amanitin, since the initial bond can be formed when aman-

itin is present but transcription cannot continue. As we notedabove, we cannot discriminate between these models from ourown results. Johnson and Chamberlin (45) showed that binarycomplexes of yeast RNA polymerase II and RNA could not onlycleave the RNA but could also add nucleotides to the newly-created 39 ends. This template-independent bond addition wascharacterized as partially sensitive to amanitin. It is difficult toenvision how amanitin functions to inhibit translocation alongthe template when it also affects bond addition in a template-independent reaction. From our own work, we can say thatamanitin does not “tie down” the active site, since amanitin-treated arrested complexes can cleave their nascent RNAs atlocations far upstream of the original polymerization site in thepresence of amanitin. However, this result could still be ob-tained if amanitin blocks downstream, but not upstream,translocation of the active site.Perhaps the most interesting aspect of our results is the fact

that amanitin does not inhibit several of the catalytic activitiesof the RNA polymerase. It is striking that amanitin inhibitionoccurs only when the active site is near the 39 end of thetranscript, with the exception of SII-mediated cleavage in ar-rested complexes. Spontaneous cleavage and pyrophosphoroly-sis in arrested complexes are both completely insensitive toamanitin. This suggests that amanitin must work through the39 end of the transcript, or alternatively, that amanitin bindsnear a location normally occupied by the 39 end of the RNA.Such an idea is consistent with the findings of Johnson andChamberlin (45), who showed that amanitin does not inhibitthe initial SII-mediated cleavage reaction in binary complexes,when the active site presumably occupies an internal positionon the transcript. However, amanitin does block any furthercleavage in these complexes. Note that after the initial cleavagein binary complexes the active site must be at the 39 end of theRNA, since bond formation can occur after the first cleavage(45). Mutations have recently been described in the largestsubunits of E. coli (46) and Bacillus subtilis (47) RNA poly-merases which confer resistance to streptolydigin. These mu-tations occur in region F (48), a segment which shows consid-erable sequence similarity among the largest subunits of botheukaryotic and prokaryotic RNA polymerases. Region F is alsothe location of amanitin resistance mutations in RNA polym-erase II (see Bartolomei and Corden (49) and references there-in), which is not unexpected since streptolydigin’s effect onprokaryotic RNA polymerase parallels the effect of amanitin onRNA polymerase II. Significantly, it has been proposed thatregion F might form part of the binding site for the 39 end of thenascent RNA (48).The fact that amanitin blocks SII-mediated transcript cleav-

age in arrested ternary complexes would seem to violate theidea that amanitin can act only near the 39 end of the tran-script. However, it is possible that amanitin simply blocksaccess of SII to the upstream sites at which transcript cleavagetakes place, rather than blocking the cleavage reaction directly.This is again consistent with the binary complex results (45).Binary complexes are probably less sterically confined thanternary complexes, and as just noted the initial SII-mediatedcleavage in binary complexes is not amanitin-sensitive.

Acknowledgments—We thank Robert Landick for SII, Zach Burtonfor the SII expression plasmid, Caroline Kane for the pGR220 U-freecassette construct, and Dave Price and Daniel Reines for communica-tion of results prior to publication and for stimulating discussions.

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Michael D. Rudd and Donal S. LuseComplexes but Fails to Inhibit Some Transcript Cleavage Modes

Amanitin Greatly Reduces the Rate of Transcription by RNA Polymerase II Ternary

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