Vol. 265, No. 1, Issue of January 5, pp. 499-505.1990 Printed in U.S.A.

Tagetitoxin: A New Inhibitor of Eukaryotic Transcription by RNA Polymerase III*

(Received for publication, May 18, 1989)

Thomas H. SteinbergzQ, Dennis E. Mathewsllll, Richard D. DurbinV, and Richard R. Burgess+ From the $Department of Oncology, McArdle Laboratory for Cancer Research and the llDepartment of Plant Pathology and Agriculture Research Service, United States Department of Agriculture, Russell Laboratories, University of Wisconsin-Madison, Madison, Wisconsin 53706

We demonstrate that tagetitoxin, a bacterial phyto- toxin, preferentially inhibits eukaryotic RNA polym- erase III. We used promoter-directed transcription of cloned genes in cell-free extracts to compare tageti- toxin inhibition of RNA polymerases from diverse sources. In HeLa cell extracts, accumulation of 5 S rRNA, and U6 snRNAs (transcribed by RNA polym- erase III) was inhibited at 0.3-3.0 fiM tagetitoxin but transcription from adenovirus 2 major late promoter (RNA polymerase II) was not significantly affected at concentrations below 30 WM. Tagetitoxin also inhibited promoter-directed RNA polymerase III transcription in cell-free extracts from Bombyx mori (pre-tRNA), and Saccharomyces cerevisiae (pre-tRNA) at 0.3-3.0 NM, concentrations that also inhibit chloroplast or bac- terial promoter-directed transcription. In nonspecific transcription assays, partially purified B. mori RNA polymerase III was inhibited by tagetitoxin at concen- trations that inhibit Escherichia coli RNA polymerase; purified calf thymus RNA polymerase II was not inhib- ited by tagetitoxin. Using injection into Xenopus laevis oocytes, we compared tagetitoxin effects on accumu- lation of Ul snRNA, hH2B mRNA (transcribed by RNA polymerase II), 5 S rRNA and U6 snRNA (RNA polym- erase III), and 5.8 S rRNA (RNA polymerase I). In Xenopus oocytes, RNA polymerase III transcription was preferentially inhibited by tagetitoxin.

Tagetitoxin, a bacterial phytotoxin that is produced by Pseudomonas syringae pv. tugetis, causes chlorosis in devel- oping but not mature plant leaves. Tagetitoxin has been purified from liquid cultures of the bacterium and a tentative structure has been assigned (Mitchell and Durbin, 1981; Mitchell et al., 1989). The biological effects of tagetitoxin can be accounted for by the failure of chloroplasts to develop in the plant apex; development of the affected plant is otherwise normal (Lukens and Durbin, 1985; Lukens et al., 1987). Tag- etitoxin inhibits RNA polymerase activity in pea chloroplast extracts and also is a potent inhibitor of Escherichia coli RNA polymerase but not of bacteriophage T7 and SP6 RNA polym- erases. Wheat germ RNA polymerase II also is insensitive to tagetitoxin (Mathews, 1988; Mathews and Durbin, 1990).

* This research was funded by National Institutes of Health Grants CA-23076, CA-07175, and CA-09135. The costs of uublication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

J To whom correspondence should be addressed. Tel.: 608-263- 3375.

11 Present address: Dept. of Agronomy and Plant Genetics, Borlang Hall, University of Minnesota, St. Paul, MN 55108.

The three recognized classes of eukaryotic nuclear RNA polymerases are distinguished by the types of genes they transcribe. RNA polymerase I transcribes rRNA genes (re- viewed in Sollner-Webb and Tower, 1986). RNA polymerase II transcribes genes encoding pre-mRNAs and most of the known small nuclear (sn) RNAs (for snRNAs, see Dahlberg and Lund, 1988). RNA polymerase III transcribes genes for 5 S rRNA, tRNAs, 7SK and 7SL RNA, U6 snRNA, and other small, stable RNAs, including several small viral RNAs (re- viewed in Geiduschek and Tocchini-Valentini, 1988). The drug cr-amanitin, a fungal octapeptide that differentially in- hibits nuclear RNA polymerases, has been invaluable in stud- ies of the functions of these eukaryotic RNA polymerases. RNA polymerase II is generally very sensitive to cY-amanitin inhibition, whereas RNA polymerase III is only moderately sensitive, and RNA polymerase I is insensitive to the inhibitor (Kedinger et al., 1970; Lindell et al., 1970; for reviews, see Chambon, 1975; Roeder, 1976; Lindell, 1980; Sentenac, 1985). We tested the effects of tagetitoxin against eukaryotic nuclear RNA polymerases from several sources and we have found that tagetitoxin, in contrast to oc-amanitin, is a selective inhibitor of RNA polymerase III. The action of tagetitoxin against RNA polymerase III promoter-directed transcription extends across a broad phylogenetic range, including verte- brates, insects, and yeast.

EXPERIMENTAL PROCEDURES

Purification oj Z’agetitoxin-The tagetitoxin used in this study was made by the method reported in Lukens and Durbin (1985). On the basis of specific activity in a bioassay and migration upon thin-layer chromatography, purity was estimated to be 70-100%. Because the purity of batches of tagetitoxin may vary, we defined a reference standard. A unit of inhibitory activity is defined here as the amount of tagetitoxin necessary for 50% inhibition of E. coli RNA polymerase activity using phage T7 as a template (see below). Tagetitoxin has an estimated molecular weight of 416 (Mitchell et al., 1989). The data presented in Fig. 3A show 50% inhibition at 0.6 gM tagetitoxin in a 50 ~1 volume (12.5 ng of toxin in 50 ~1) so the specific activity of the preparation we used for these studies was about 80,000 units/mg.

We have recently been testing tagetitoxin preparations available from a commercial source (Epicentre Technologies, Madison, WI), and we have observed activity against E. coli RNA polymerase and against promoter-directed RNA polymerase III transcription in Xen- opus S-150 extracts.’

Plasmids-The plasmid pSmaF, containing Ad2 MLP,’ originally cloned in the laboratory of R. G. Roeder (The Rockefeller University, New York, NY), was obtained from P. Farnham, University of Wis- consin-Madison. The plasmid pGEM5S contains a Xenopus borealis somatic 5 S RNA maxigene (Xbs + 20, Sakonju et al., 1980), originally provided by D. Brown, Carnegie Institute of Washington, Baltimore,

1 T. Steinberg and H. Paaren, unpublished results. * The abbreviations used are: Ad2 MLP, adenovirus 2 major late

promoter; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.

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MD, subcloned into the Hind111 site of the vector pCEM1 (Promega Biotec). This subclone of the 5 S maxigene was from the laboratory of J. E. Dahlberg, University of Wisconsin-Madison. The plasmid pmU6 contains a 707-bp BumHI-Hind111 fragment of a m&se U6 snRNA gene cloned into Bluescript vector (Stratagene. San Diego. CA) to obtain pmU6 -315/+286; this plasmid, obtained from E. L&d (University of Wisconsin, Madison, WI), was a gift from R. Reddy, Baylor College of Medicine, Houston, TX.

The plasmid pWTALA5, containing 437-base pair silkworm (Bom- byn mori) tRNA*‘” gene (Larson et al., 1983) was a gift from Connie White and Karen Sprague, University of Oregon, Eugene OR. The plasmid pT7-8+, cbnt&ning a yea& (Sacc~arornyc~s cereuisiue) tRNAPro gene (Cummins et al.. 1985) was a gift from James Hendrick and Michael Culbertson, University of Wisconsin-Madison.

The plasmid pGEMhH2b contains the human histone H2b se- quences -162 to +630 relative to the mRNA cap site, inserted into the pGEM-3Zf(+) vector (Promega). The original human genomic H2b clone (from which this construct was made), obtained from E. Lund, was a gift from H. Sive and R. Roeder (The Rockefeller University). The hH2b gene is accurately transcribed by RNA polym- erase II when this plasmid is injected into Xenopus oocytes (Thomp- son et al., 1989). The.plasmid pBi33 contains a human Ul snRNA maxigene (Skuzeski et al., 1984).

Extracts-Hela cell nuclear extracts were prepared by a modifica- tion of the method of Dignam et al. (1983) as described in Thompson et al. (1989). HeLa S-100 extracts were prepared by the method of Weil et al. (1979). All HeLa cell extracts were made from log phase cells.

Posterior silkgland extract from B. mori (Wilson et al., 1985) was a gift from Connie White and Karen Sprague, University of Oregon, Eugene, Oregon. Yeast extract (Engelke et al., 1985) was a gift from James Hendrick and Michael Culbertson, University of Wisconsin- Madison.

Transcription Conditions-Promoter-directed transcription in cell- free extracts was in 25-~1 volumes. Nucleotide concentrations were 400 pM except for GTP, which was 20 gM. Label was 10 &i of [a-3’P]GTP (Du Pont-New England Nuclear). a-Amanitin was from Sigma. For all eukaryotic in vitro transcriptions, components were mixed together on ice before transcription was initiated by addition of nucleotides and label. HeLa cell extracts were incubated at 30 “C; silkworm and yeast extracts were incubated at 24 “C. Reactions were terminated after 30 min by addition of 100 ~1 of 0.5 mg/ml proteinase K, 0.5% sodium dodecyl sulfate, 5 mM EDTA, 50 mM Tris, pH 7.4, and then incubated at 37 “C for 15 min before extraction of RNA with phenol/chloroform. For Ad2 MLP, final salt and buffer concen- trations were: 10 mM Hepes (pH 7.9). 50 mM KCl, 7.5 mM MgCl~, 10% glycerol, and 0.5 mti dithiothreitol; 6 ~1 of nuclear extract-and 300 ng of gel-purified SmaI insert from PSmaF plasmid was used per react&n. heia S-100 transcription conditions-were as reported-by Weil et al. (1979) except that 5% glycerol was included in each reaction. DNA concentrations were: 5 S maxigenes, 1.2 pg/reaction and for U6,1.6 pg/reaction. Silkworm extract transcription conditions were as reported in Wilson et al. (1985). Yeast extract transcription conditions were as in Engelke et al. (1985). For all RNA polymerase III promoter-directed assays, DNA was added as supercoilkd plasmid.

Purified RNA Polvmerases-For all RNA polvmerase assavs. re- action bolumes were”50 ~1 with 1 PCi of [3H]GTfi (ICN Radio&em- icals). Reaction components were mixed on ice and transcription initiated by addition of nucleotides. Reactions were incubated at 30 ‘C for 20 min before they were stopped with 5 ~1 of 10% sodium dodecyl sulfate. A portion (45 ~1) was applied to DEAE-cellulose discs. The filters were air-dried, washed 5 times with a solution of 5% (w/v) anhydrous dibasic sodium phosphate, washed twice with double-distilled water and twice with 95% ethanol. Filters were dried and scintillation counted.

E. coli RNA polymerase holoenzyme, provided by Dayle Hager, University of Wisconsin-Madison, was made by a modification of the method of Burgess and Jendrisak (1975). For transcription on T7 DNA, 300 ng of holoenzyme and 2.25 fig of T7 DNA were used per reaction. Transcription buffer, salt, and nucleotide concentration conditions were as recommended by Chamberlin et al. (1979). The T7 DNA was from Sigma. For nonspecific transcriptions, 500 ng of holoenzvme and 7.5 11a of denatured calf thvmus DNA (Worthintion) was use2 per react&n; nucleotide concentrations were: 500 pM ATP, GTP, and CTP, and 20 yM UTP.

Calf thymus RNA polymerase II, provided by Dallas Aronson, University of Wisconsin-Madison, was purified by the method of Hodo and Blatti (1977). Transcription was on denatured calf thymus

DNA in 25 mM Tris-HCl (pH 7.9), 8 mM MgClZ, 25 mM (NH&Sod, 500 PM ATP, GTP, CTP, 20 FM UTP, with 140 ng of RNA polym- erase, and 7.5 pg of DNA/reaction.

Partially purified B. mori RNA polymerase III, a gift from Lisa Young and Karen Sprague (University of Oregon, Eugene, OR), was made by a modification of the procedure in Ottonello et al. (1988). The material (a fast protein liquid chromatography fraction of the P- 450 pool) we used in our assays contains transcription factor IIIB but was purified away from the other RNA polymerase III transcription factors and from the cu-amanitin-sensitive RNA polymerase II activity in the crude extract. Transcription was on denatured calf thymus DNA in 50 mM Tris-HCl, 150 mM KCl, 2 mM MnCl*, 1 mM dithio- threitol, 500 /IM ATP, GTP, CTP, 20 yM UTP, with 5 ~1 of RNA polymerase III fraction, and 7.5 pg of DNA/reaction.

Oocyte injections-Oocyte injections were done and the RNA re- covered as described in Thompson et al. (1989). Oocytes were incu- bated at 18 “C for 14-24 h; in any experiment, the incubation time of the various samples was the same. Obcytes were harvested (pooled or individually) bv disruption in proteinase K solution. digested at 37 “C for 1 h, an: toial nucleic acids extracted.

I

Electrophoresk and Autoradiography-Electrophoresis was on 42 X 30 cm X 0.6 mm, 7.5% (30:0.8), cross-linked polyacrylamide gels with 8 M urea and Tris borate buffer. The DNA markers included in every electrophoresis were the end-labeled products of an MspI digest of pBR322; the markers visible on the gels were 622, 527, 404, 309, 238/242, 217, 201, 190, 180, 160, 147, 123, 110, 90, 76, and 67 bases long. Sizes of some of the visible markers are indicated in the figures. Gels were run at 45 or 50 watts constant power (1350-1700 volts) for about 3 h. Autoradiography was done with Kodak XAR-5 film at -80 “C without intensifying screens. For densitometry, exposures with the linear range of the film were scanned using a LKB soft laser scanning densitometer (Biomed Instruments, Chicago, IL).

RESULTS

Human RNA Polymerase II Is Relatively Resistant to Tag- etitorin-HeLa cell nuclear extracts were tested for their sensitivity to tagetitoxin. When RNA polymerase II transcrip- tion from the Ad2 MLP was assayed using the pSmaF tem- plate, no significant inhibition was noted below lo-30 PM tagetitoxin (Fig. lA). Transcription of the run-off RNA occurs at significant levels at up to 100 pM and is readily detected at 300 FM tagetitoxin.

HeLa S-100 Promoter-directed RNA Polymerose III Tran- scription Is Sensitive to Tagetitoxin-Tagetitoxin sensitivity of RNA polymerase III activity in HeLk S-100 extracts was tested by using several diffbrent genes known to be transcribed by RNA polymerase III (class III genes). The X. borealis somatic 5 S rRNA maxigene is efficiently and accurately transcribed in HeLa S-100 extracts. As shown in Fig. lB, transcription of 5 S RNA is clearly reduced at about 1 pM

tagetitoxin and is eliminated in the lo-30 pM range. As expected, transcription of this gene is resistant to 1 rg/ml a- amanitin but is virtually eliminated by 100 pg/ml cu-amanitin, as is typical of the a-amanitin response of vertebrate RNA polymerase III transcribed genes.

The U6 snRNA gene is a member of a recently described class of RNA polymerase III-transcribed genes with pro- moters external to the coding region of the gene (Krol et al., 1987; Das et al., 1988; reviewed by Folk, 1988 and Sollner- Webb, 1988). Fig. 1C shows that inhibition of U6 transcription by tagetitoxin in the S-100 extracts is similar to that of 5 S.

We tested tagetitoxin effects on transcription of other genes known to be transcribed by RNA polymerase III in S-100 extracts. Transcription of the Epstein-Barr virus EBER RNAs, also called the EBV J RNAs (Arrand and Rymo, 1982), was inhibited by tagetitoxin, as was transcription of cloned murine tRNA genes (data not shown).

S-150 extracts from X. luevis oocytes are particularly active in RNA polymerase III transcription of the somatic 5 S genes (Millstein et aZ., 1987). Transcription of the 5 S maxigene in Xenopus S-150 extracts is very sensitive to tagetitoxin with

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FIG. 1. Transcription by RNA polymerase II and RNA po- lvmerase III in HeLa cell extracts with increasing levels of tagetitoxin. Autoradiograms of RNA after electrophorests in a 7.5% polyacrylamide, 8 M urea denaturing gel are shown. A, transcription by RNA polymerase II. The transcription template was the SmaI fragment bearing the Ad2 MLP from the plasmid pSmaF. Lane I, no inhibitor added, lane 2, 1 rg/ml n-amanitin; lanes 3-8, increasing levels of tagetitoxin: 1.0, 3.0, 10, 30, 100, 300 pM, respectively, and as indicated above. R, RNA polymerase III 5 S maxigene transcription. M, DNA markers (see “Experimental Procedures”); lane 1, no inhib- itor added; lane 2, 1 rg/ml Lu-amanitin; lane 3, 100 /*g/ml n-amanitin; lanes 4-8, increasing levels of tagetitoxin: 0.3, 1.0, 3.0, 10, 30 NM tagetitoxin, respectively. C, U6 RNA transcription. The U6 RNA products are indicated by an arrow. 44, DNA markers; lane 1, no inhibitor; lane 2, 1 pg/ml ol-amanitin; lane 3, 100 pg/ml cu-amanitin; lanes 4-8, increasing levels of tagetitoxin: 0.3, 1.0, 3.0, 10, 30 pM, respectively.

RNA accumulation markedly reduced at 0.3-1.0 PM and abol- ished at 10 PM tagetitoxin (data not shown). In the S-150 extracts, transcription of the U6 gene is inhibited by tageti- toxin to about the same degree as in HeLa S-100 extracts (data not shown).

Silkworm tRNA Transcription Is Sensitive to Tagetitoxin- Crude extracts from B. mori posterior silk glands accurately and efficiently transcribe tRNA genes (Wilson et al., 1985). Transcription of a tRNA*‘” gene in the presence of cr-amanitin or tagetitoxin (Fig. 2A) illustrates the unusual resistance of insect RNA polymerase III to relatively high (100 pg/ml) levels of a-amanitin and also indicates that tagetitoxin is a potent inhibitor of promoter-directed B. mori RNA polymer- ase III transcription. The small differential sensitivity of the

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yeast extracts. Autoradiograms of RNA after electrophoresis in polyacrylamide-urea gels are shown. A, transcription of H. mori tRNAsA’” in a posterior silk gland extract. M, DNA markers; lane I, no inhibitor; lane 2,1 pg/ml tw-amanitin; lane 3, 100 Fg/ml cu-amanitin; lanes 4-9, increasing levels of tagetitoxin: 0.1, 6.3, 1.0, 3.0, IO, 30 pM, respectively. The primary transcript is marked by an arrow; the larger RNA bands are due to readthrough and downstream termination. B, transcription of S. cereuisiae tRNA’“’ in a yeast extract. M, DNA markers; lane I, no inhibitor; lanes 2-6, increasing levels of tageti- toxin: 0.1, 0.3, 1.0, 3.0, 10 pM, respectively. An arrow indicates the primary transcript.

larger readthrough band versus the major transcript is a consistent result in our hands. Transcription of a Drosophila melanogaster tRNA gene in a crude D. melanogaster larval extract is similarly resistant to a-amanitin and sensitive to tagetitoxin.”

Yeast tRNA Transcription Is Sensitive to Tagetitoxin- Yeast RNA polymerase III is also resistant to high levels of a-amanitin (Sentenac, 1985). Transcription of a yeast tRNAPro gene in a yeast extract (Fig. 2B) is sensitive to tagetitoxin at levels that inhibit RNA polymerase III tran- scription in vertebrate and insect extracts.

Tagetitorin Inhibits E. coli RNA Polymerase and B. mori RNA Polymerase III but Not Calf Thymus RNA Polymerase II-Details of tagetitoxin inhibition of chloroplast and bac- terial RNA polymerase transcription are presented elsewhere (Mathews, 1988; Mathews and Durbin, 1990). As a compari- son standard we measured the response of E. coli RNA polym- erase holoenzyme to increasing levels of tagetitoxin, using phage T7 DNA as template. The results obtained with T7

” T. Steinberg and D. Jackson, unpublished results.

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502 Tagetitoxin Inhibits RNA Polymerase III

DNA (Fig. 3A) are in agreement with previous work using phage T4 DNA as a template showing 50% inhibition at the 0.3-1.0 pM concentration range. Inhibition is virtually com- plete at lo-30 FM tagetitoxin.

As further confirmation that tagetitoxin acts against RNA polymerase III rather than a transcription co-factor, we tested nonspecific transcription of some purified RNA polymerases using denatured calf thymus DNA as the template. Under these circumstances, E. coli RNA polymerase is also inhibited by tagetitoxin, but about 3-fold more toxin is required for a corresponding degree of inhibition as on T7 DNA (Fig. 3A). Purified calf thymus RNA polymerase II transcribing calf thymus DNA is resistant to tagetitoxin but 3. mori (silkworm) RNA polymerase III is inhibited by tagetitoxin to about the same degree as E. coli RNA polymerase on calf thymus DNA (Fig. 3A).

Tagetitoxin Inhibition of Promoter-directed Transcription by RNA Polymerase III Is Quantitatively Similar to Inhibition of E. coli RNA Polymeruse-To determine relative RNA ac- cumulation at different tagetitoxin concentrations, autoradi-

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FIG. 3. Quantitative comparison of tagetitoxin inhibition in vitro. A, RNA polymerase activity in the presence of increasing levels of tagetitoxin. E. coli holoenzyme was incubated with wild type phase T7 DNA (Eco RNAP 7’7) or with denatured calf thvmus DNA iE& RNAP CT), as indicated. Purified calf thymus RNA polymerase II was incubated with calf thvmus DNA (CT RNAP II). as was partially purified silkworm RNA polymer&e III (SW RNAP III). Accumulation of total RNA is expressed as percent of the positive control (no tagetitoxin added). Duplicate samples were assayed at each concentration; the range in values (counts/min) at any point was less than 2%. Transcription conditions were as indicated under “Experimental Procedures.” B, tagetitoxin effects on eukaryotic pro- moter-directed transcription in extracts. Activity is expressed as percent of the untreated control. Films were exposed without inten- sifying screens for appropriate times so that the film exposure and densitometry was in the linear range or bands were excised and scintillation counted. HeLu, HeLa cell extracts. Ad2 MLP transcrip- tion was measured using nuclear extracts; 5 S and U6 transcription was measured using S-100 extracts. Y, yeast extract; SW, silkworm extract. For A and B, tagetitoxin concentration is expressed in a logarithmic scale so the r-axis is discontinuous.

ograms corresponding to Figs. 1, A, B, C, 2, A and B, or replicates of these experiments were scanned in a densitom- eter or the bands excised and counted by scintillation. Tage- titoxin inhibition of RNA polymerase III promoter-directed transcription is quantitatively similar to the response of E. coli RNA polymerase. Transcription of the 5 S rRNA maxi- gene in HeLa extracts and of silkworm and yeast pre-tRNA genes in homologus extracts is 50% of the control at about 0.3-1.0 pM (Fig. 3B). Transcription of the 5 S maxigene in Xenopus extracts is inhibited to the same degree (data not shown). This is comparable to the concentration of tagetitoxin required to inhibit E. coli RNA polymerase using phage T7 or T4 DNA as a template or to inhibit transcription of chloro- plast tRNA genes in chloroplast extracts (Mathews and Dur- bin, 1990).

In vitro transcription of the U6 snRNA gene is also sensitive to tagetitoxin but a higher concentration is required to attain the same degree of inhibition as for the 5 S genes in both HeLa (Fig. 3B) and Xenopus (data not shown) extracts. In contrast, RNA polymerase II promoter-directed transcription, as measured in HeLa cell nuclear extracts using the Ad2 major late promoter, is relatively resistant to tagetitoxin.

Differential Inhibition of RNA Polymerases in Injected Oo- cytes-A convenient way to monitor the in vivo activity of the eukaryotic nuclear RNA polymerases on cloned genes is by injection of various templates into X. Levis oocytes (Gurdon and Wickens, 1983). By coinjecting labeled nucleotides, tran- scription of endogenous oocyte genes also can be examined. Xenopus oocytes offer the advantage that all three RNA polymerases are simultaneously active and their activity can be compared in a single injection. By oocyte injection we can assay the in uiuo effects of small amounts of tagetitoxin without regard to permeability barriers. Injection of tageti- toxin into the oocyte cytoplasm or nucleus selectively inhibits accumulation of endogenous oocyte RNA.” We have tested the effect of a single dose of tagetitoxin coinjected with several different genes.

The human histone H2b gene, like the histone genes of sea urchins (Probst et al., 1979) and X. luevis (Heindl et al., 1988), is accurately transcribed and processed to yield a stable mRNA when injected into Xenopus oocytes. Fig. 4A (lanes 4- 6) shows the results of coinjection of a human H2b gene and the 5 S maxigene with either 15 pg of a-amanitin/oocyte (lane 5) or 650 pg of tagetitoxin/oocyte (lane 4). A single injection of about 650 pg of tagetitoxin is sufficient to abolish transcrip- tion from the 5 S maxigene, while transcription from the H2b gene is about half of the control. Fig. 4A (lanes 1-3) shows the results of coinjection of the mouse U6 gene and the 5 S maxigene with tagetitoxin or a-amanitin. Transcription of the U6 gene and the 5 S maxigene is resistant to 15 pg of a- amanitin (lane 2) but sensitive to 650 pg of tagetitoxin (lane 1).

Human Ul snRNA genes injected into Xenopus oocytes are efficiently and accurately transcribed by RNA polymerase II (Murphy et al., 1982, 1987a; Skuzeski et al., 1984; for review, Dahlberg and Lund, 1988). Fig. 4B shows results from coin- jection into oocytes of a human histone (H2b) gene, a human Ul maxigene, a 5 S maxigene, and a mouse U6 gene with 520 pg of tagetitoxin. Transcription from the histone gene and the Ul maxigene is not significantly inhibited while transcrip- tion from the 5 S maxigene and the U6 gene is decreased.

To further explore the relative responses of the oocyte RNA polymerases to tagetitoxin we coinjected into oocytes a mix- ture of the Ul maxigene, the 5 S maxigene and the U6 gene with different amounts of tagetitoxin. Fig. 4C shows the

’ E. Lund and T. Steinberg, unpublished results.

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FIG. 4. Coinjection of several genes with tagetitoxin into X. laevis oocytes. Autoradiograms of RNA alter electrophoresis in polyacrylamide-urea gels. A, coinjection of hH2b and 5 S maxigene or 5 S maxigene and U6. M, DNA markers; lanes Z-3,5 S maxigene and U6; lanes 4-6, hH2b and 5 S maxigene. Lanes 2 and ?, 650 pg of tagetitoxin; lanes 2 and 5, 15 pg of tu-amanitin; lanes 3 and 6, positive control. Injection volume was 20 nl; DNA levels: 1 ng of hH2b and 5 S maxigene and 0.3 ng of UG/oocyte. Label: 0.05 PCi of [cu-,“P]GTP/oocyte. About 15 oocytes/treatment were injected. Incubation was for 14 h at 18 ‘C. Oocytes were pooled and the total RNA extracted. One-half oocyte equivalent of RNA was loaded per lane. B, coinjection of hH2b, Ul maxigene, 5 S maxigene, and U6. Lane I, no tagetitoxin; lane 2, 520 pg of tagetitoxin. M, DNA markers. Injection volume was 20 nl; DNA levels: 1 ng of hH2b, 1 ng of Ul maxigene, 0.25 ng of 5 S maxigene, and 0.15 ng of U6 per oocyte. Label: 0.20 PCi of [n-“P]GTP/oocyte. 10 oocytes were pooled per sample and the RNA extracted; also four individual oocytes were extracted per sample. The individual oocytes gave RNA profiles similar to the corresponding pooled samples. Oocytes were incubated for 23 h. One-half oocyte equivalent of RNA was loaded per lane. C, coinjection of Ul maxigene, 5 S maxigene, and U6 with varying levels of tagetitoxin. M, DNA markers. Lane 1, no Inhibitor; lane 2, 30 pg of o-amanitin/oocyte; lanes 3-7, increasing amounts of tagetitoxin per oocyte; lane 3, 50 pg; lane 4, 260 pg; lane 5, 1.3 ng; lane 6, 4.3 ng; lane 7, 13 ng of tagetitoxin. DNA levels: 2 ng Ul maxigene, 0.2 ng of 5 S maxigene, and 0.3 ng of UG/oocyte. Label: 0.25 &i of [cu-‘“P]GTP/oocyte. At least 30 oocytes were injected per sample; 25-30 oocytes were pooled and the RNA extracted. Three individual oocytes from each sample were also extracted; The RNA profiles from the individual oocytes were identical to those of the pooled samples for each treatment. Oocytes were incubated for 20 h. One-half oocyte equivalent of RNA was loaded per lane.

response of such a mixture with increasing levels of tageti- toxin. The extreme sensitivity to tagetitoxin of 5 S transcrip- tion correlates well with in vitro assays using Xenopus oocyte S-150 extracts (data not shown) and with the other oocyte injections presented above. Transcription of U6 RNA is more sensitive to tagetitoxin than transcription of Ul RNA and can be virtually abolished at concentrations of tagetitoxin that allow significant levels of Ul transcription, as is evident from Fig. 4C, lanes 6 and 7; at such tagetitoxin concentrations the predominant U6 transcript is several nucleotides longer than in the control. This residual transcript does not appear to be the result of RNA polymerase II transcription.* U6 transcription does not appear to be as sensitive to tagetitoxin as 5 S; at the lowest amount injected, 50 pg/oocyte (Fig. 4C, lane 3), there is a slight increase in U6 transcription while 5 S transcription is depressed. At high tagetitoxin concentra- tions there is a sharp decline in U6 transcription. Comparison of 5 S and U6 transcription in Fig. 4.4, lanes l-3, and Fig. 4B also indicates that U6 transcription is less sensitive to tage- titoxin than 5 S transcription. As expected, Ul maxigene transcription is sensitive to relatively low levels of cu-amanitin but resistant to tagetitoxin inhibition at levels sufficient to abolish 5 S transcription and severely depress U6 transcrip- tion.

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0 0 0 03 01 0 32 10 32 10 32

Tagetrtoxln, ng Injected

FIG. 5. Quantitative comparisons of the response of injected genes and endogenous 5.8 S transcription to tagetitoxin. Den- sitometry of appropriate exposures of autoradlograms corresponding to Fig. 4C was done with a soft laser densitometer. Activities are expressed as percent of untreated control. Tagetitoxin concentrations are expressed in a logarithmic scale, so the x-axis is discontinuous. Assuming the volume of an oocyte to be 1.0 ~1, the 1.0 ng of tagetitoxin injected results in an average concentration of 2.3 PM.

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504 Tagetitoxin Inhibits RNA Polymerase III

We measured the relative responses of the injected genes to tagetitoxin by densitometry of autoradiograms correspond- ing to Fig. 4C and also determined the accumulation of endogenous X. laevis 5.8 S RNA, a product of RNA polym- erase I transcription. Fig. 5 shows relative accumulations in response to varying tagetitoxin levels of the Ul, 5 S, and U6 RNAs (from injected genes) and the endogenous 5.8 S RNAs. If the volume of an oocyte is taken as 1.0 ~1, then injection of 1.0 ng of tagetitoxin gives a concentration of about 2.3 FM. The patterns of Ul and 5 S transcription inhibition by tage- titoxin are consistant with data from in vitro transcriptions; the in vivo assays give quantitatively similar results. Inhibi- tion of U6 transcription is similar to inhibition of 5 S tran- scription, but requires higher concentrations of tagetitoxin. The different tagetitoxin levels required for 5 S and U6 RNA transcription inhibition in oocytes resembles the situation for the in vitro transcriptions using cell-free extracts. As can be seen from Fig. 5 and as also may be inferred from Fig. 4C, X. laeuis RNA polymerase I appears to be insensitive to tageti- toxin, with slight inhibition at the higher levels.

DISCUSSION

We have shown that tagetitoxin, recently shown to have activity against chloroplast and bacterial RNA polymerases, can selectively inhibit RNA polymerase III in vitro and in Xenopus oocytes. Inhibition of RNA polymerase III was both specific and severe; transcription of 5 S RNA and tRNA genes in all extracts that were tested was inhibited by levels of tagetitoxin similar to that required for the corresponding inhibition of E. coli RNA polymerase, using phage T7 DNA as a template.

Eukaryotic nuclear RNA polymerases probably arose by gene duplication and subsequent divergence (Sentenac, 1985). Comparisons of nucleotide sequences and derived amino acid sequences from cloned genes for the largest subunits of yeast RNA polymerases I (Memet et al., 1988a), II and III (Allison et al., 1985) show extensive regions of homology to the E. coli @’ subunit. The second largest subunit of Drosophila RNA polymerase II shows extensive homology to E. coli RNA polymerase /3 subunit and to a chloroplast DNA open reading frame (Falkenburget al., 1987). Tagetitoxin inhibition of RNA polymerase III transcription in promoter-directed assays closely resembles that of E. coli and chloroplast RNA polym- erases. The inhibition by tagetitoxin of promoter-directed RNA polymerase III transcription from a wide range of or- ganism (vertebrates, insects, and yeast) is striking; by con- trast, there is a wide range of sensitivity of RNA polymerase III to cr-amanitin. There may be in RNA polymerase III a conserved structure to which tagetitoxin binds; it is not yet clear if tagetitoxin inhibition of the bacterial and eukaryotic RNA polymerases results from binding to homologous re- gions.

The largest subunit of vaccinia virus RNA polymerase, a multisubunit virally encoded enzyme, has extensive regions of homology to the largest subunits of eukaryotic RNA polym- erases II and III and the p’ subunit of E. coli RNA polymerase. (Broyles and Moss, 1986). Transcription by purified vaccinia RNA polymerase is not inhibited by high levels of or-amanitin or rifampicin (Baroudy and Moss, 1980; Spencer et al., 1980). We have not yet tested tagetitoxin for inhibitory activity against vaccinia RNA polymerase.

On the basis of the inhibition of silkworm RNA polymerase III in nonspecific transcription (Fig. 3A) we believe that tagetitoxin acts directly against RNA polymerase III, rather than by binding a transcription factor. We do not yet have explanations for the sensitivity difference (&IO-fold) to tag-

etitoxin of E. coli RNA polymerase holoenzyme and of silk- worm RNA polymerase III in nonspecific transcription on calf thymus DNA uersus promoter-directed transcription. Tagetitoxin response of RNA polymerase may depend on different enzyme conformations that vary according to the template or protein cofactors in the transcription complexes. It may be that cycles of re-initiation in the promoter-directed RNA polymerase III transcriptions exaggerate the inhibitory effect of a given tagetitoxin concentration. We will continue to investigate these matters using purified enzymes and de- fined templates.

It is not clear why our assays show a difference in tageti- toxin inhibition of U6 transcription in comparison to 5 S transcription. The U6 genes (and also the 7SK RNA genes) differ from other genes that are transcribed by RNA polym- erase III in the location and identity of cis-acting promoter elements (Krol et al., 198’7; Das et al., 1988; Murphy et al., 1987b; reviewed by Folk, 1988 and Sollner-Webb, 1988). Some of the transcription factors that are required for in vitro transcription of U6 RNA are not the same as those required for 5 S RNA or tRNA (Reddy, 1988). We are extending our in vitro studies to other templates to try to determine if there is a consistent pattern in tagetitoxin inhibition of classes of RNA polymerase III transcribed genes. Further in vitro and in uiuo studies may also help settle questions of relative tagetitoxin sensitivity for transcription of different class III genes.

Tagetitoxin does not affect plant development, other than causing chlorosis by preventing chloroplast development. Pre- sumably in uiuo plant nuclear RNA polymerases are relatively unaffected by tagetitoxin. Preliminary experiments using pu- rified wheat germ RNA polymerase III in nonspecific tran- scription assays indicate that the plant enzyme is at least lo- fold less sensitive to tagetitoxin than B. mori RNA polymerase III.’ Wheat germ RNA polymerase II is not inhibited by tagetitoxin concentrations below 100 pM (Mathews and Dur- bin, 1990) and partially purified wheat germ RNA polymerase I is similarly insensitive to tagetitoxin inhibition.5 The ac- tivities of Xenopw and human RNA polymerases have so far been measured using promoter-directed assays in crude ex- tracts. Comparisons of the tagetitoxin inhibition of fraction- ated and purified Xenopus and wheat germ RNA polymerases will be helpful in clarifying the relative sensitivity of nuclear RNA polymerases.

On the basis of the response of endogenous Xenopus oocyte transcription, RNA polymerase I does not seem to be affected by tagetitoxin. Further studies will be required to better describe RNA polymerase I sensitivity. Sequence comparisons of conserved domains of the largest subunits of the three yeast RNA polymerases indicate that RNA polymerases II and III are more closely related to each other than to RNA polymer- ase I (Memet et al., 1988b). It may be that the action of the two inhibitors of eukaryotic RNA polymerases, cY-amanitin and tagetitoxin, acting against RNA polymerase II and III but not against RNA polymerase I, reflects conservation of binding sites between RNA polymerases II and III.

Selective inhibitors of transcription by eukaryotic RNA polymerases are not common. Tagetitoxin is the first example of a specific RNA polymerase inhibitor that acts against bacterial RNA polymerases and against only one (RNA po- lymerase III) of the eukaryotic nuclear RNA polymerases. According to the available structural data (Mitchell et al., 1989), tagetitoxin represents a new class of RNA polymerase inhibitors. We expect that further analyses of the action of tagetitoxin on eukaryotic and prokaryotic RNA polymerases

5 T. Steinberg and J. Jendrisak, unpublished data.

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Tagetitoxin Inhibits RNA Polymerase III 505

will be useful in dissecting transcription mechanisms.

Acknowledgments-We thank Nancy E. Thompson for valuable discussion, Dayle Hager for purified E. coli RNA polymerase holo- enzyme, and Dallas Aronson for purified calf thymus RNA polymer- ase II. We are very grateful to Connie White, Lisa Young, and Karen Sprague for gifts of silkworm extracts and RNA polymerase III and for sharing unuublished data. We thank James Hendrick and Michael Culbertson for providing yeast extract and for their interest in this work. We thank Jerry Jendrisak and Herb Paaren of Epicentre Technologies for providing wheat germ RNA polymerases and tage- titoxin samples and for sharing unpublished data. We thank David Jackson and Michael Hoffman for sharing unpublished data. We thank Peggy Farnham for critical reading of the manuscript, Jody Flatt for providing HeLa cells, Lynda Schilling for sharing unpub- lished data, and other members of the Burgess and Farnham labora- tories for helpful comments. We are especially indebted to James E. Dahlberg and Elsebet Lund for use of oocyte injection facilities, collaboration, encouragement, and critical reading of the manuscript.

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T H Steinberg, D E Mathews, R D Durbin and R R BurgessTagetitoxin: a new inhibitor of eukaryotic transcription by RNA polymerase III.

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