Properties of a Drosophila RNA Polymerase I1 Elongation Factor*

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May 25, 2015 - of GTP, ATP, and UTP, 30 p~ CTP, 50-100 mM KCl, 0.2-0.4 pCi ... Reactions were generally .... but RNA chains are less than 20 nucleotides in length. The ... longer for the RNA polymerase I1 alone, the amount of labeled.
Vol. 264, No. 15,Issue of May 25,pp. 8963-8969,

1989 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Properties of a Drosophila RNA Polymerase I1 Elongation Factor* (Received for publication, November 23, 1988)

Ann E. SluderS, Arno L. Greenleaf, and David H. Prices From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

W e have purified from nuclear extracts of Drosophila Kc cells a 36-kDa protein, DmS-11, which has an effect on the elongation properties of RNApolymerase 11. DmS-I1 stimulates RNA polymerase I1 during the transcription of double-strandedDNA templates when the nonphysiological divalent cation manganese is present. In the presence of physiological mono- and divalent cations, the factor reduces the tendency of RNA polymerase I1 to pause at specific sites along a dC-tailed template including the major pause encountered after 14 nucleotides have been incorporated. Based on its size and chromatographic properties, as well as itsability to stimulate RNA polymerase I1 activity in the presence of manganese, the protein seems to be analogous to a factor S-I1 purified from mouse cells (Sekimizu, K., Kobayashi, N., Mizuno, D., and Natori, S. (1976) Biochemistry 15, 5064-5070). We have used a completely defined system and show that the properties of DmS-I1 are intrinsic to the factor and not mediated through other factors.

RNA polymerase I1 sequentially utilizes a number of accessory factors during the multistep process of transcription. Biochemical studies have demonstrated the existence of distinct intermediates in the formation of active transcription complexes (Fire et al., 1984; Hawley and Roeder, 1985; Van Dyke et al., 1988). Recent evidence suggests that the control of elongation is responsible for regulating the expression of several genes (Bender et al., 1987; Kao et al. 1987; Rougvie and Lis, 1988). The production of an elongation-competent complex has been examined using crude extracts (Cai and Luse, 1987, a and b; Luse et al., 1987) or partially purified factors (Van Dyke et al., 1988). However, many factors are required to enable RNA polymerase I1 to initiate accurately at a promoter and a completely defined RNA polymerase I1 i n vitro transcription system has not been developed. The inability to obtain defined elongation complexes with purified RNA polymerase I1 and specific factors has hampered efforts to study the elongation phase of transcription. This problem can be circumvented by using an assay that utilizes a dCtailed double-stranded template on which purified RNA polymerase I1 willinitiate at a specific site (Kadesch and Chamberlin, 1982; Sluder et al., 1988). An understanding of the requirements for efficient elongation is a necessary prelude to * This work wassupported by National Institutes of Health Grants GM35500 and GM28078. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. $. Present address: Dept. of Genetics, Harvard Medical School and Dept. of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114. Present address: Dept. of Biochemistry, University of Iowa, Iowa City, IA 52242.

the i n vitro study of the control of transcription by pausing and/or termination. A number of factors have been identified which affect the elongation and termination properties of procaryotic RNA polymerase (reviewed in Yager and von Hippel, 1987; Chamberlin et al., 1985; Horwitz et al., 1987). Only a few factors which affect the elongation properties of RNA polymerase I1 have been identified. RNase H has been shown to stimulate elongation i n vitro (Sekeris et al., 1972; Kane, 1988; Sluder et al., 1988).A number of RNA polymerase I1 stimulatory factors have been reported (see Sekimizu et al., 1979 or Revie and Dahmus, 1979 and references therein). In some cases, these stimulatory factors were shown to have an effect on elongation (Spindler, 1979; Rappaport et al., 1987; Reinberg and Roeder, 1987). Factor 5 from Drosophila K, cells affects elongation (Price et al., 1987) and is also required for accurate initiation i n vitro (Price et al., 1989). AnRNA polymerase I1 stimulatory factor, S-11, initially purified from mouse cells (Sekimizu et al., 1976) has been shown to increase the efficiency of elongation of RNA polymerase I1 (Rappaport et al., 1987; Reinberg and Roeder, 1987). This factor was first detected by its ability to stimulate RNA synthesis by RNA polymerase I1 in the presence of nonphysiological divalent cation manganese (Sekimizu et al., 1976). In thepresence of magnesium, S-I1 seems to allow the polymerase to pass through specific sequences more efficiently than it would without the factor (Rappaport et al., 1987; Reinberg and Roeder, 1987). This ability of S-I1 to attenuate pausing of RNA polymerase I1 has not been demonstrated with purified factors and thus has not been shown to be an intrinsic property of S-11. Wereport here the purification of an activity from Drosophila Kc cell nuclear extracts that is capable of affecting RNA polymerase I1 elongation in a completely defined system. This Drosophila factor, which we call DmS-11, reduces the time spent by RNA polymerase I1 at a subset of the numerous pause sites encountered on a dC-tailed template. EXPERIMENTALPROCEDURES

Materials-All materials were reagent grade or as described in Sluder et al. (1988). Templates-The two templates used in these studies were prepared as described by Sluder et al., 1988. dC-BalI-E contains the BalI-E fragment of adenovirus 2 cloned in pBR322 linearized with PstI and tailed with dCTP andterminal transferase at both ends (Coulter and Greenleaf, 1985). dC-3025 is a 250-base pair PstI/EcoRI fragment containing the 3’-end of Drosophila histone H3 tailed at the PstIend (Price and Parker, 1984). RNA Polymerase II Elongation Assays-Unless otherwise indicated, the final conditions in individual assays were identical with those described in Sluder et al., 1988: 20 mM HEPES’ (diluted from 500 mM HEPES, pH 7.8), 5 mM MgCl,, 600 p M concentration each of GTP, ATP, and UTP, 30 p~ CTP, 50-100 mM KCl, 0.2-0.4 pCi of [ ~ u - ~ ’ P ] C T Pof / ~reaction I volume; DNA template, purified DroThe abbreviation used is: HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.

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sophila RNA polymerase I1 (Price et aL, 1987), and purified DmS-I1 as indicated in individual experiments. Reactions were generally started by the addition of RNA polymerase I1 to a mixture of the buffer, substrates, cofactors, and template. Any other factors were usually added later. For some reactions, UTP was omitted from the start mixture. Low levels of UTP present in the other nucleotides supported limited transcription. After a 5-10-min incubation under these UTP-limiting conditions, UTP was added to 600 PM. In pulse/ chase experiments, labeling was accomplished under normal transcription conditions with CTP being added to a final concentration of 1.2 mM during the chase period. Reactions were stopped with 150 pi of a Sarkosyl solution (1% Sarkosyl, 100 mM NaCl, 100 mM Tris, pH 8.0, 10 mM EDTA, and 100 pg/ml tRNA), and the nucleic acids were isolated as detailed in Sluder et al. (1988). Reaction products were analyzed in polyacrylamide, 6 M urea, TBE (89 mM Trizma base, 89 mM boric acid, 2 mM EDTA) gels as described (Price et al., 1987). Assays for stimulation of RNA polymerase I1by DmS-I1 were adapted from Nakanishi etal. (1981). Reactions were similar to those described above except that MgC12 was replaced by MnC12 at a final concentration of 2 mM in the individual reactions andthe KC1 concentration was 20-25 mM. RNA polymerase I1 was added to a large initial reaction mixture that was incubated for 1 min before being aliquoted into tubes containing the fractions to be assayed. Individual assays were incubated for 15 min before being stopped, spotted onto DE81 filters, and counted as described in Price et al. (1987). Stimulation was calculated by dividing the totalcounts incorporated in each reaction by the amount incorporated by RNA polymerase I1 alone. Chromatography-Conditions and buffers used during purification of DmS-11, as well as the preparation of Kc cell nuclear extracts, are described in Price et al. (1987). Details of the purification are described in the text. Densitometry-RNA distributions were determined by scanning the autoradiograph with a Zeineh soft laser scanning densitometer (Biomed Instruments). Maximum lengths were determined from the leading edge of the distribution RNA species (defined as theboundary marking the top 5% of the density). Average lengths were determined from the median of the RNA distributions. This method of defining RNA sizes slightly overestimates transcript length in the early time points, especially for the median lengths, since a portion of the transcripts will have migrated off the bottom of the gel and thuswill not be considered in the measurement.

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FIG. 1. Purification of DmS-11. A combination of ion exchange and hydrophobic interaction chromatography was used to purify DmS-I1 from Drosophiia K, cell nuclear extracts. Vertical and sianted lines indicate step and gradient elutions, respectively. Elutions were carried out with KC1 unless otherwise indicated. Conditions for chromatography are described in the text and under “Experimental Procedures.”

onto a200-ml phosphocellulose (P-11)column equilibrated in 125 mM KC1. This column was step-eluted as indicated, and, after dialysis, the P-11-0.4 M step (40 mgof protein) was loaded onto a 30-ml DEAE-cellulose (DE52) column in 125 mMKC1. The flow-through (20 mg of protein) was dialyzed and loaded onto a 1-ml Mono Q column equilibrated in 25 mM KCI. Solid (NH4)2S04was added to the Mono Q-FT (5 mg of protein) to a final concentration of 1 M. The fraction was centrifuged (5000 rpm for 10 min in a Sorvall HB-4 rotor) to remove a very slight precipitate, and thenwas loaded onto RESULTS a 1-ml Pharmacia LKB Biotechnology Inc. phenyl-Superose Purification of DmS-ZZ-We have fractionated nuclear ex- fast protein liquid chromatography column equilibrated in 1 tracts from Drosophila Kc cells and identified three activities M (NH4)2S04.Proteins were eluted with a salt gradient dethat affect the elongation properties of RNA polymerase I1 creasing from l M to 0 M (NH4)2S04. Thefractions eluted (Price et al., 1987; Sluder et al., 1988; Price et al., 1989). The from this column contained ammonium ions, and thus could elongation assay we employed utilizes a double-stranded, dC- not be accurately assayed for stimulatory activity (see Sluder tailed template (Kadesch and Chamberlin, 1982) on which et al., 1988). Fractions that contained DmS-I1 were identified pure RNA polymerase I1 will initiate three nucleotides up- by analysis on a silver-stained sodium dodecyl sulfate protein stream of the single strand/double strand junction (Sluder et gel (data not shown). Preliminary purification schemes had al., 1988). When the crude extracts were chromatographed on allowed the tentative identification of a 36-kDa protein as phosphocellulose (P-11), thefraction containingproteins that DmS-11. This 36-kDa protein eluted from the phenyl-Superelute between 0.3 M and 0.4 M KC1 (P-11-0.4 M step) dramat- ose column between 740 mM and 540 mM (NH&S04. These ically stimulated RNA polymerase I1 in this assay (Price et fractions, which contained 150 pg of protein, were pooled,and al., 1987). Further chromatography of this fraction resulted the salt concentration was adjusted to 100 mM KC1 by a in the separation of three factors, each of which had a char- combination of dialysis and dilution. The material was then acteristic influence on elongating RNA polymerase I1 (Price loaded onto a 1-ml Mono S column in 100 mMKC1, and et al., 1987; Sluder et al., 1988; Price et al., 1989; and results proteins were eluted with a 100 mM to 350 mM KC1 gradient. presented here). Inorder to determine if any of the Drosophila Fig. 2A shows the DmS-I1 activity profile of the Mono S stimulatory factors corresponded to the RNA polymerase I1 column. The fractions with DmS-I1 activity contained apstimulatory factor S-I1 (Sekimizu et al.,1976), the conditions proximately 5 pg of protein. Sodium dodecyl sulfate-protein of the dC-tailed template reactions were adapted to those gel analysis indicated that the 36-kDa protein constituted optimal for S-I1 stimulation(Nakanishi et al., 1981). The greater than 95% of the protein in fractions 33 and 34, which most notable change was the replacement of M 8 + by Mn2+ also contained the peak of activity (Fig. 2B). The presence of (see “Experimental Procedures”). DmS-I1 activity is defined other stimulatoryactivities and otherpolymerases in the early here as the stimulation of RNA polymerase I1 transcription stages of purification, as well as the presence of ammonium ions after the phenyl-Superose column made it impossible to of a dC-tailed template under these modified conditions. The purification of DmS-I1 is summarized in Fig. 1. The quantitate the amount of DmS-I1 in various fractions and Kc cell nuclear extract (1.6 g of protein in 65 ml) was loaded impossible to calculate a fold purification. A fold purification

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analyzed on a 6% polyacrylamide TBE/urea gel (Fig. 3). RNA polymerase I1 alone pauses for extended periods of time at numerous sites along the template as indicated by the presence of specific bands on thegel. Three effects of DmS-I1 can -0 be seen in Fig. 3. First, even though the pulse time was 50% 3 longer forthe RNA polymerase I1 alone, the amountof labeled .E 1.4 200 B m RNA was greater if DmS-I1 was present. This will be adE 150 dressed below. Second, some, but not all,of the RNA species c produced by pausing a t specific sites were greatly reduced in 1.o 100 the presence of DmS-11. The RNA of about 383 nucleotides 30 31 32 33 34 35 36 37 (Fig. 3, arrowhead) is much less apparent when DmS-I1 was fraction number present in the reaction. Third, the average elongation rate was increased by DmS-I1 as indicated by the average density of transcripts shifting up the gel in the presence of DmS-11. B 3031 32 33 34353637 We have seen in this and other experiments not shown that the maximum elongation rate determined from the leading edge of the transcript density is stimulated by DmS-I1 only slightly (less than 2-fold), but the average elongation rate is stimulated 3-4-fold once thepolymerase has passed the major pulse a t nucleotide 383. kDa One of the interesting features of the transcriptionof a dCtailed templateby RNA polymerase I1 is the strong pause site 66encountered after 14 nucleotides have been incorporated into RNA (Sluder etal., 1988). Since DmS-I1 reduced the pausing 45 a t some sites during the transcription of alongdC-tailed template, we wanted to determine if it had anyeffect on the pausing of the 14-mer. The fact thatmore RNA was synthe29 sized in thepresence of DmS-I1 in thepulse chase experiment just described indicated thatDmS-I1 might increase the number of RNA polymerase I1 molecules that were productively engaged in the synthesis of long transcripts, which would be FIG.2. Chromatography of DmS-I1 on Mono S . Mono S was used as thefinal column in the purification of DmS-11. A, 1p1 of each expected for factors that reduce pausing at the 14-mer. The effect of DmS-I1 on the 14-mer was addressed in two of the 0.5-ml fractions eluting from the column was assayed for the ability to stimulate transcription on the dC-RalI-E template by 45 or related experiments.Fig. 4 shows the effect of DmS-I1 on the 90 units of RNA polymerase I1 in the presence of MnCL (reported relative production of the 14-nucleotide RNA and full-length fold stimulation was theaverage of the two determinations). Conduc- runoff transcript on the 245-base pair fragment of dC-tailed tivity measurements were taken and used to calculate the concentra- aDm3025 (dC-3025). An increase in runoff transcript corretion of KC1 in the fractions.R, the indicated fractions were analyzed on a 6-15% gradient polyacrylamide, SDS protein gel; proteins were lated with a decrease in the amountof 14-mer as theconcen-

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of about 100,000 can be estimated if it is assumed that the overall yield of DmS-I1 was 33% (80% a t each column). DmS-II SuppressesPausing-Although DmS-11-like activities require manganese in order for RNA polymerase I1 to exhibit the full stimulatory response (Sekimizu et al., 1976), the factor does have an effect on magnesium. Other workers have found that mammalian S-I1 has an effect on the elongation properties of RNA polymerase I1 that has initiated from the adenovirus major late promoter in uitro (Rappaport et al., 1987; Reinberg and Roeder, 1987). Their results indicated thatS-I1 facilitated the appearance of full length runoff transcript by reducing pausing a t a small number of specific sites, although theeffect was not demonstrated to be a direct effect ofS-11. Purified DmS-I1 was tested for its effect on purified Drosophila RNApolymerase I1 using a dC-tailed template assay in the presenceof magnesium. A pulse/chase protocol was followed in which RNA polymerase I1 was first preincubated with the dC-BalI-E template under conditions of limiting UTP (Sluder etal., 1988). Under these conditions, polymerase binds to the template and begins RNA synthesis, but RNA chains are less than 20 nucleotides in length. The reaction was split into two tubes, both of which contained normal levels of UTP, butonly one of which contained DmS11. After 3 min of synthesis (2 min if DmS-I1 was present), the labeled nucleotide was diluted with cold CTP, and chase timepoints were takenat various times.TheRNA was

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FIG. 3. Effect of DmS-I1 on the elongation properties of RNA polymerase 11. A 6% polyacrylamide RNA gel was used to analyze the RNA synthesized by RNA polymerase I1 alone (- lanes) or in the presence of DmS-I1 (+ lanes). Two 25-pl reactions were set up, each containing 112 units of RNA polymerase 11, 90 mM KCI, 5 mM MgC12, and 10 pg/ml dC-RalI-Eunder pulseconditions. One reaction also contained 50 ng of DmS-I1 (Mono S fraction 34 from Fig. 2). After a 3-min pulse (2 min if DmS-I1 was present), the chase was initiated with the addition of CTP (1.2 mM final concentration) and 3-pl aliquots were removed and stopped a t the indicated chase times. The reaction conditions for the dC-tailed template assays can he found under "Experimental Procedures." The RNA was less than 300 nucleotides long at the beginning of the chase and was analyzed in a 6% polyacrylamide TBE/urea gel. The migration of DNA size standards is indicated, and thearrowhead denotes the migration of a 383-nucleotide RNA species.

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KCI, 5 mM MgCI2, and 0.5 pg/mldC-3025 template. Individual reactions contained the indicated volume of DmS-I1 (Mono S fraction 34 from Fig. 2). which had a proteinconcentration of 5 pg/ml. Continuous labeling reactions wereperformed as described under “Experimental Procedures”; reaction time was 10min. The RNA was analyzed in an 18% polyacrylamide TBE/urea gel. The small arrowhead indicates the 14-mer; full length runoff is indicated by the large arrowhead. R, the sections of each lane containing the 14-mer and the full length runoff transcript were excised from the driedgel. The gel slices were placed in scintillation fluid and counted. In order to obtain a relative measure of the moles of the two RNA species, the counts were normalizedby dividing by the numberof cytidine residues in the RNA (3for the 14-mer, 62 for the runoff).

FIG.5. Time course of the effect of DmS-I1 on RNA polymerase I1 pausing at the 14-mer site. A pulse-chase protocol was followed in which RNA polymerase I1 (320 units) was preincubated for 5 min with 25 pg of dC-3025 template in a volume of 16 pl. A labeling mixture (11 pl) containing nucleotides, MgC12, and [CY-~~P] CTP was then added, and incubation was continued for 5 min; the concentrations of all reaction components were 2 X that of standard in vitro transcription reactions (see “Experimental Procedures”). A t theend of this5-min pulsing incubation, a 1.75-pl aliquot was removed to a tube containing100 pl of ice-cold Sarkosyl solutionwith 100pg/ml tRNA;this wasdesignated the zero timepoint.The remainder of the reactionwasdivided into two chasereactions containing 1.2 mM unlabeled CTP. One chasereaction was performed without DmS-I1 ( A ) and the other ( B ) contained 40 ng of DmS-I1 (Mono S fraction 34 from Fig. 2). The final reaction volumes were 25 pl; the KC1 concentration was maintained a t 100 mM throughout the reaction series. At the indicated times, 3-pl aliquots were removed and stopped asdescribed for the zero time point. The purified RNA was analyzed in an 18% polyacrylamide gel; only that region of the gel containing the 14-17-nucleotide RNAs is shown. C, the levels of 14-mer. whichis the predominant band in each lane, werequantitated by scintillation counting of excised gel slices. The counts were normalized using thezero time point as the100% value.

legend to Fig. 5 for details of the procedure). DmS-I1 was found to increase the rate a t which RNA polymerase I1 left the 14-mer pause site by approximately 10-fold (Fig. 5). In no experiment was 100% of the 14-mer chased into longer RNA tration of DmS-I1 was increased,suggesting that DmS-I1 reduced the time that thepolymerase was paused at the 14- species. The level of 14-mer that appeared resistant to chasnucleotide site. In the presence of DmS-11, some polymerase ing, 20% in Fig. 5, varied somewhatfromexperimentto molecules first paused at 13 rather than 14nucleotides (Fig. experiment. At least a portion of this residual 14-mer is due 4).* When the DmS-I1 levels used were higher than those in to dissociation of the 14-mer from the polymerase and template a t long reaction times (Sluder etal., 1988). The relation Fig. 4, the small RNAs becameevenmoreheterogeneous, ranging from 10 to 17 nucleotides in size (data not shown). between the amountof 14-mer not elongated during the chase The smaller (i.e. 10-13-nucleotide) RNAs were not produced and the amount released from the ternary complex was not if DmS-I1 was added to pulse-labeled RNA during a chase examined in detail. Drosophila ElongationFactors HaveAdditive Effects-Each and therefore were not degradation products of longer RNA of the three elongation factors we have identified stimulate species (Fig. 3.’ A pulse-chase protocol was used to determine more directlythe elongation rate of RNA polymerase I1 in vitro (Price et if DmS-I1 decreased the residence time of RNA polymerase al., 1987; Sluder et al., 1988; Price et al., 1989). RNase H acts I1 at the 14-mer pause site. RNA transcripts were initiated by allowing renaturation of the template during transcription and partially elongated in an initial reaction containing [a- which has about a 2-fold stimulatory effect on theelongation :’2P]CTP;the lengthof time for this pulse reaction waschosen rate (Sluder etal., 1988). Factor 5 does not affect the location so that themajor product would be the 14-mer. The reaction of the many pause sites encounteredby RNA polymerase 11, was diluted intoa mixture containingexcess unlabeled CTP, but has aneffect of decreasing the lengthof time thatpolymet 1989). We have examined erase pausesat the sites (Price al., and incubation was continuedeitherwithorwithoutthe addition of DmS-11. Aliquots were removed at various times, the effect of all possible combinations of the three factors on and the amount of 14-mer remaining was determined (see the elongation properties of RNA polymerase 11. Fig. 6 shows the results of a pulse-chase experiment in which factor 5 or A. E. Sluder, A. L. Greenleaf, and D. H. Price, unpublishedresults. factor 5 and DmS-I1 are present in thereactions. The condi-

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FIG.6. Effect of DmS-I1 in the presence of factor 5. A, a pulse-chase protocol was used to examine the elongation properties of RNA polymerase I1 (65 units) in the presenceof 72 ng of factor 5 or 72 ng of factor 5 and 10 ng of DmS-11. Pulse labeling was for 5 min under limiting U T P concentrations (see “Experimental Procedures”) in the presence of the factors. Excess cold C T P was added, and chases were performed a t 50 mM KC1 for the indicated times. RNA was analyzed on a 6% polyacrylamide TBE/urea gel. The lane marked M contains DNA size standards (1631, 998, 633, 512, 396, 344, 298, 220, and 154 nucleotides). The arrowhead indicates the migration of a 383-nucleotide RNA species. B, the maximum and average RNA length at each time pointwas determined as described under “ExperimentalProcedures,” and the results were plotted uersus the chasetime. tions of the reactionswere similar to those inFig. 3. Factor 5 stimulated theelongation rate about5-fold alone, but did not change the pattern of pause sites usually found with pure RNA polymerase I1 (Fig. 6). When DmS-I1was present with factor 5, there was a dramatic reduction of the pause sites (Fig. 6). In addition to thereduction of the pausing, DmS-I1 also further stimulated the elongate rate about 2-fold. In the presence of bothfactors,the polymerase molecules transcribed the dC-tailed template inarelatively synchronous manner. DmS-II Is Not Stably Bound to the Elongation ComplexI t has been reported that mammalian S-I1 is associated with RNA polymerase I1 in ternary complexes (Horikoshi et al., 1984). In preliminary experiments,we found thatDmS-I1 did RNA polymerase I1 during not stably associate with pure glycerol gradient sedimentation.* In order tosee if the factor was associated with the elongation complex, a dilution experiment was devised. Ternary complexes were generated with or without DmS-I1 such that the nascentRNA was less than 300 nucleotides in length. The chains were lengthened under chase conditions after diluting the reactions up to 27-fold. Fig. 7 indicates that the characteristic suppression of pausing

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FIG. 7. Elongation complex dilution. Two 25-pI reactions were set up, each containing 75 units of RNA polymerase 11, 90 mM KCI, pulse conditions 5 mM MgC12, and 10pg/ml dC-BalI-E template under either with or without 40 ng of DmS-11. After a 3-min pulse (2 min if DmS-I1 was present), the reactions were diluted either 0-, 3-, 9-, or 27-fold, keeping all other reaction conditions equal and chased for 30 min. Details of the reactions are under “Experimental Procedures.” The presence of DmS-I1 is indicated by a + over the appropriate lanes. The RNA was analyzed on a 6% polyacrylamide TBE/urea gel. The arrowhead indicates the mobility of a 383-nucleotide RNA species.

is reduced by diluting the complexes. Likewise, the stimula: tion of elongation rate is reduced by dilution. The pulse took place under concentrated conditions in which DmS-I1 could suppresspausing at the 14-mersiteand,thus,thelanes containing the factor show the expectedincrease in RNA synthesis. These results indicate that DmS-I1 is not stably bound to the ternarycomplex and, thus, mustbe recruited at individual pause sites. We have obtained similar resultswith factor 5 in dilution experiments (Price et al., 1989). It has been difficult to conclusively determine if elongation complexes formed in the presence of both factor 5 and DmS-I1 are stable to dilution because of the difference in magnitude of the effects of the twofactors. In several unpublished experiments in which elongation complexes were physically separated from free factors by gel exclusion chromatography, no DmS-I1 could be found with thecomplexes if factor 5 was included in the reaction. DISCUSSION

Elongation by RNA polymerase I1 is a poorly understood process. RNA polymerase I1 has anin vivo elongation rate of greater than1,000 nucleotidesper min (Ucker and Yamamoto, 1984) and canproduce transcripts over 100,000 nucleotides in length. In contrast, theaverage rate of elongation in vitro on double-stranded templates is a t least 20-fold slower when physiological mono- and divalent cations areused. Since it is very difficult to interpret experiments concerningelongation and termination in vivo, we have worked toward obtaining a meaningful, defined in vitro system in which the elongation properties of RNA polymerase I1 and specific factors that modulate these properties can be studied. Toward this end, we have identified three activities in K, cell nuclear extracts that modify the elongation properties of RNA polymerase I1 on dC-tailed, double-stranded templates. Here we have described one factor,DmS-11, a 36-kDa protein which suppresses

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Drosophila RNA Polymerase I f Elongation Factor

pausing by RNA polymerase I1 at a limited number of pause sites. It is likely that DmS-I1 is the Drosophila analog of mammalian $11, as the molecular weight and chromatographic properties of DmS-I1 are similar to those of the mouse (Sekimizu et al., 1976) and calf thymus (Rappaport et al., 1987) proteins. A molecular weight has not been reported for HeLa S-11, but it also exhibits similar chromatographic behavior (Reinberg and Roeder, 1987). The stimulatory activity of mouse S-I1 has been reported to require manganese (Nakanishi et al., 1981). When assayed with the dC-tailed template, DmS-I1 will stimulate RNA polymerase I1 elongation in the presence of magnesium (Fig. 3). This difference from mouse S-I1 could represent true species variation or may reflect the difference in templates used since the mouse factor was assayed with total genomic DNA as template. HeLa S-I1 has also been found to stimulate RNA polymerase I1 transcription of a dC-tailed template in the absence of manganese (Reinberg and Roeder, 1987). Two additional chromatographic species of S-I1 stimulatory activity have been reported from mouse cells (Sekimizu et al., 1976; Nakanishi et al., 1981) and calf thymus (Rappaport et al., 1987). For the mouse factor, these other species were found to be phosphorylated (Sekimizu et al., 1981) and proteolyzed (Horikoshi et al., 1985) forms of S11. To date, only a single form of DmS-I1 has been observed. The molecular weight suggests that thisrepresents the unproteolyzed form; the phosphorylation state of the Drosophila protein is not known. Both mouse and HeLa S-I1 willcosediment with RNA polymerase I1 during glycerol gradient centrifugation (Horikoshi et al., 1984; Reinberg and Roeder, 1987), and thus appear to interact with polymerase in the absence of transcription. Mouse S-I1 has also been shown to be associated with the ternary complex of polymerase, template, and transcript during nonspecific transcription (Horikoshi et al., 1984). In similar glycerol gradient sedimentation experiments, DmS-I1 interacted only weakly with RNA polymerase I1 (data not shown). We have demonstrated here that DmS-I1 is not stably associated with RNA polymerase I1 during elongation on the dC-tailed templates. However, it remains to be determined if these differences are significant or merely reflect different experimental conditions (e.g. the use of KC1 rather than (NH4)2S04 and use the of magnesium rather than manganese). We have consistently observed that high levels of DmS-I1 inhibit RNA polymerase I1 activity in the dC-tailed template/manganese assay (data not shown), but have not determined whether the inhibition occurs during initiation orelongation. This inhibition has notbeen reported for the mammalian factors. S-I1 is probably involved in specific transcription. Antibodies raised against S-I1 from mouse Erhlich ascites tumor cells inhibit specific transcription in HeLa wholecell extracts (Sekimizu et al., 1982). S-I1 isolated from HeLa extracts (Reinberg and Roeder, 1987) or calf thymus (Rappaportet al., 1987) does not seem to be required for specific initiation, but has been shown to stimulate the appearance of full length runoff transcripts during specific transcription from the adenovirus major late promoter. Our results indicate that one role of DmS-I1 is to stimulate the elongation reaction, although we cannot rule out any involvement in specific initiation. Wehave shown here that the ability of DmS-I1 to suppress pausing is an intrinsic property of the factor and polymerase and is not due to an indirect effect of the factor on other factors present in the previously reported experiments with mammalian S-11. DmS-I1 appears to reduce the variation in elongation rate among individual polymerase molecules, as indicated by the

decreased heterogeneity in the sizes of RNA produced during a chase reaction (Fig. 3 and 6). The major pause sites are the main determinants of overall elongation rate in the dC-tailed templates. If the only effect of DmS-I1 was to suppress pausing, then it should significantly stimulate the elongation rate. While there is some stimulation of maximum elongation rate, most of the effect is seen on the average rate. DmS-11increases the elongation rate through some sequences, but appears to decrease the rate of others (Figs. 3 and 7). The pausing induced by DmS-I1 at other sites at least partially explains the low level of rate stimulation, although the enhancedpause sites do not appear to be as strong as the ones which are suppressed. It is possible that DmS-I1 may slow one or more of the steps of the elongation cycle, thus counteracting the stimulatory effects of suppression of pausing. This latter possibility could be addressed by determining the effect of DmS-11 on the rate of RNA polymerase I1 elongation on a template such a poly[d(A-T)]where pausing is minimal. A number of comparisons can be made between DmS-I1 and theEscherichia coli elongation control proteinnus A. nus A interacts with RNA polymerase in a sequence-dependent manner during elongation, becoming associated with the polymerase following the release of u factor (Greenblatt and Li, 1981; Greenblatt et al., 1987). Purified nus A reduces the elongation rate of E. coli RNA polymerase (Schmidtand Chamberlin, 1984a) and enhances polymerase pausing at some sites (Kassavetis andChamberlin, 1981; Farnham et al., 1982;Landick and Yanofsky, 1984).DmS-I1affects elongation similarly in that italters the behavior of RNA polymerase I1 at some but not allpause sites and may in some way slow the elongation cycle. Unlike nus A , however, DmS-I1 is observed to suppress as well as enhance pausing and, at least on the dC-BalI-E template, suppression is the more dramatic effect. nus A protein is a required component of the elongation control complex whichmediates antitermination of transcription by phage X N protein (Horwitz et al., 1987) and may also mediate interactions between RNA polymerase and the termination factor p (Schmidtand Chamberlin, 1984b). The interaction of nus A with elongating polymerase seems to involve rapid association and dissociation rather than stable binding (Schmidt andChamberlin, 1984a),but theassociation is apparently stabilized by the assembly of a complete elongation control complex (Greenblatt et al., 1987; Horwitz et al., 1987). Similarly, DmS-I1 is not stably associated with RNA polymerase I1 during elongation, although stable association of mouse S-I1 with elongating polymerase under different reaction conditions has been reported (Horikoshi et al., 1984). If DmS-I1 is in fact a nus A-like elongation control factor, then it may serve to mediate the effect of other regulatory factors on RNA polymerase I1 elongation and termination. These additional factors could perhaps be detected in vitro by a stabilization of the association of DmS-I1 with elongating polymerase or by an alteration of the effect of DmS-I1 on elongation at particular sites on the template. Recently, the sequence of a cDNA clone of the mouse S-I1 was reported, but no homology was found with other proteins, including nus A (Hirashima et al., 1988). REFERENCES Bender, T. P., Thompson, C. B., and Kuehl, W. M. (1987) Science 237,1473-1476 Cai, H., and Luse, D. S. (1987a) J. Bwl. Chem. 262,298-304 Cai, H., and Luse, D. S. (1987b) Mol. Cell. Bwl. 7,3371-3379 Chamberlin, M. J., Briat, J.-F., Dedrick, R. L., Hanna, M., Kane, c. M., Levin, J., Reynolds, R., and Schmidt, M. (1985) in Genetics, Cell Differentiation, and Cancer (Marks, P., ed) pp. 47-73, Academic Press, Orlando, FL

Drosophila RNA Polymerase II Elongation Factor Coulter, D. E., and Greenleaf, A. L. (1985) J. Biol. Chem. 260,1319013198 Farnham, P. J.,Greenblatt, J., and Platt, T.(1982) Cell 2 9 , 945-951 Fire, A., Samuels, M., and Sharp, P. A. (1984) J. Biol. Chem. 259, 2509-2516 Greenblatt, J., and Li, J. (1981) Cell 24, 421-428 Greenblatt, J., Horwitz, R. J., and Li, J. (1987) in RNA Polymerase and the Regulation of Transcription (Reznikoff, W. S., Burgess, R. R., Dahlberg, J. E., Gross, C. A., Record, M. T., Jr., and Wickens, M. P., eds) pp. 357-366, Elsevier Science Publishing Co., Inc., New York Hawley, D. K., and Roeder, R. G. (1985) J. Biol. Chem. 260, 81638172 Hirashima, S., Hirai, H., Nakanishi, Y., and Natori, S. (1988) J. Biol. Chem. 263,3858-3863 Horikoshi, M., Sekimizu, K., and Natori, S. (1984) J. Biol. Chem. 259,608-611 Horikoshi, M., Sekimizu, K., Hirashima, S., Mitsuhashi, Y., and Natori, S. (1985) J. Biol. Chem. 260,5739-5744 Horwitz, R.J., Li, J., and Greenblatt, J. (1987) Cell 5 1 , 631-641 Kadesch, T. R., and Chamberlin, M. J. (1982) J. Biol. Chem. 267, 5286-5295 Kane, C. M. (1988) Biochemistry 27,3187-3196 Kao, S.-Y., Calman, A. F., Luciw, P. A., and Peterlin, B. M. (1987) Nature 330, 489-493 Kassavetis, G. A., and Chamberlin, M. J. (1981) J. Biol. Chem. 2 5 6 , 2777-2786 Landick, R., and Yanofsky, C. (1984) J . Biol. Chem. 259, 1155011555 Luse, D. S., Kochel, T., Keumpel, E. D., Coppola, J. A., and Cai, H. (1987) J. Biol. Chem. 262,289-297 Morrissey, J. H. (1981) Anal. Biochem. 117, 307-310 Nakanishi, Y., Mitsuhashi, Y., Sekimizu, K., Yokoi, H., Tanaka, Y., Horikoshi, M., and Natori, S. (1981) FEBS Lett. 1 3 0 , 69-72 Price, D. H., and Parker, C. S. (1984) Cell 3 8 , 423-429

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