Functional steps in transcription initiation and reinitiation from the ...

TKEJOURNALOF BIOLOGICAL CHEMISTRY 0 1987 by The American Society of Biological Chemists, Inc.

Vol. 262, No. 8, Issue of March 15, pp. 3452-3461 1987 Printed in &SA.

Functional Steps in Transcription Initiation and Reinitiation from the Major Late Promoter in a HeLa Nuclear Extract* (Received for publication, September 18,1986)

Diane K. Hawley4 and RobertG. Roederg Fronz The Rockefeller University, Laboratory of Biochemistry and Molecular Biology,New York, New York 10021

Transcription initiationby RNA polymerase I1 from was inhibited from producing a transcript if Sarkosyl was theadenovirus major late promoter was studied in added to a concentration of 0.05% before the addition of vitro in a nuclear extract derived from HeLa cells. We nucleotides. We argued that the step necessary to confer found that different concentrations of Sarkosyl resistance to 0.05% Sarkosyl was probably formation of the blockedtranscriptioninitiationatseveraldifferent first phosphodiester bond, because only the appropriate comfunctional steps. These correspondedto two steps de- bination of two nucleotides, those corresponding to the first fined previously in a partially purified transcription two nucieotides incorporated into the RNA, would result in system: formation of the rapid start complex, which conversion from Sarkosyl sensitivity to Sarkosyl resistance. required incubation of template with the extract and However, we will refer to this step as productive initiation to was blocked by Sarkosyl concentrations >0.025%, and include the possibility that elongation beyond the first phosproductive initiation, which occurredupon addition of nucleotides andwas prevented by Sarkosyl concentra- phodiester bond was required to convert the rapid start comtions >0.1%. We used assays based on these findings to plex to an elongation complex capable of completing a transcript in the presence of concentrations of Sarkosyl>O.O5%. probe further the events that comprise the processes of The RNA polymerase I1 transcription system we used preinitiation and reinitiation of tran~riptionfromthe major late promoter. We found that the templates used viously wasreconstituted with protein fractions prepared from in the first round of initiations from preformed rapid HeLa cell nuclear extracts by chromato~aphyon phosphostart complexes were moreactively transcribed in the cellulose. Wechose to use a partially purified system initially to eliminate possible complexities resulting from the presence secondroundof initiations than newly addedtemplates. We argued that the most likely explanation for of endogenous nucleotides. In this study, we have tested the this observation was that some transcription compo- applicability of these methods to unfractionated nuclear exnent(s) remained committedto the promoterafter for- tracts. mation of an elongation complex. This template-com- There were two main incentives for developing the methmitted complex must have been distinct from the rapid odology necessary for defining discrete functional steps in the startcomplex,becausereinitiationandrapidstart initiation mechanism in a transcription system reconstituted complex formationwere both blocked by0.025% Sar- from an unfractionated extract. First,most other laboratories kosyl. studying RNA polymerase I1 transcription in general or the expression in vitro of a specific class I1 promoter use nuclear or whole cell extracts. Yet many aspects of these transcription systems have never been adequately characterized. Most of We previously demonstrated that transcription initiation the effort directed toward optimizing conditions in vitro has from the adenovirus major late promoter could be separated focused on obtaining the maximum signal or the greatest intothree functional steps in vitro (I). Thesesteps were difference in signal without an understanding of what reaction defined on the basis of successive changes in the sensitivity parameters have been affected by the change in reaction of the initiation reaction to Sarkosyl upon incubation of the conditions. Such an approach can result in uncertainty about promoter with the protein fractions required to reconstitute whether the observed behavior in vitro is of physiological transcription. For example, an incubation at 30 “C in the relevance. An increased knowledge about the nuclear extract absence of nucleotides was both necessary and sufficient to system will therefore be useful both to those wishing to use confer resistance to Sarkosyl concentrations in the range of nuclear extracts in vitro and also to those seeking to interpret 0.0~5-0.02%.We called the intermediate formed during this published information obtained using nuclear extracts. Secincubation the “rapid start complex” because it was capable ond, it has been demonstrated that transcription from the of initiating transcription within 30 s after addition of nu- major late promoter requires at least five proteins in addition cleoside triphosphates. Once formed, the rapid start complex to RNA polymerase I1 and that other promoters use additional factors not required by the major late promoter (2-12). The * This research was supported by Grants CA 42567 and CA 34223 ability to obtain mechanistic informat~onin the unfractionfrom the National Institutes of Health (to R. G. R.) and with general ated system, even if this information is quite limited, could support from the Pew Trusts. The costs of publication of this article were defrayed in part by the payment of page charges. This article be valuable in assessing whether all of the factors involved in must therefore be hereby marked “advertisement” in accordance with transcribing a particular promoter in vitro are present in a transcription system reconstituted from purified components. 18 U.S.C. Section 1734 solely to indicate this fact. $ Supported by a postdoctoral fellowship from the National InstiIn this paper, we describe our analysis of functional steps tutes of Health. Present address: University of Oregon, Institute of in transcription initiation from the major late promoter in Molecular Biology, Eugene, OR 9’7403. nuclear extract. We have found conditions 3 To whom correspondence should be addressed: The Rockefeller the unfractiona~d University, Laboratory of Biochemistry and Molecular Biology, 1230 that allow measurement of the rate and extentof reinitiation of transcription and have used this method to show that York Ave., New York, NY 10021.

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Steps in I n i t ~ t i o nand R e ~ n ~ t ~by t wRNA n P o l y ~ II~ ~ e

and the amount of transcript produced in each reaction was measured following gel electrophoresis of the reaction products. The second addition of Sarkosyl was included in the experimental protocoi to prevent reinitiation from occurring in all reactions, even those in which the amount of Sarkosyl already present in the reaction was not sufficiently high to limit transcription to a single round of initiations. This method simplified interpretation of the data and allowed a direct determination of the concentrations of Sarkosyl required to inhibit formation of the rapid start complex (curue a) and productive initiation of transcription (curve b). The concentrations of Sarkosyl required for inhibition of both steps in nuclear extracts were somewhat higher than those required in the fractionated system. For example, complete inhibition of rapid start complex formation required 0.025%Sarkosyl in X SARKOSYL the nuclear extract, but only 0.015-0.02% in the fractionated system. Similarly, when Sarkosyl was added to the reaction after rapid start complex formation, the amount of Sarkosyl required for complete inhibition was shifted from 0.05% in the fractionated system to 0.1% in thenuclear extract. We previously reported that concentrations of Sarkosyl greater than or equal to 0.05%, when added after nucleotides, FIG. 2. Concentrations of Sarkosyl required to inhibit two interfered with production of full-length transcripts from the steps in transcription initiationin uibro. SarkosyI was added to major late promoter and caused the a~cumulationof tranindividual transcription reactions at two different times, indicated by the lettersa and b. For each reaction, major late promoter-containing scripts that were prematurely paused or terminated about 186 plasmid DNA (pMLH2) was incubated with nuclear extract for 60 nucleotides downstream of the cap site (1).This behavior was min at 30 “C in a total volume of 20 al, using the standard conditions also observed with the nuclear extract, although the effective described under “Experimental Procedures.”For the a reactions (0), concentration of Sarkosyl was found to be shifted to around Sarkosyl a t the Concentrations shown was present during the initial 0.1% (data not shown). To eliminate this complexity, we have incubation. For the b reactions (O), Sarkosylwasaddedto the s constructed a plasmid containing the major late promoter but indicated concentrations following the initial incubation and 30 before additionof nucleoside triphosphates (NTP’s).Additional Sar- lacking the downstream sequences we have found to be rekosyl was added to all reactions to a final concentration of between quired for the production of the prematurely terminated tran0.08% and 0.1% 30 s after nucleotide addition. The reactions, in a scripts (see “Experimental Procedures;” Fig. 1).This plasmid, final volume of 30 &l, were incubated at 30 “C for an additional 45 pMLH2, was the template used in most of the experiments min. The transcription reactions were processed and subjected to 2. The producpolyacrylamide gel electrophoresisas described under “Experimental described in this paper, including that of Fig. tion of a f u l l ~ e n ~ 306-nucleotide h transcript from this temProcedures.”The3%-nucleotiderun-offtranscriptswereexcised from the gel and the amountof transcript was determined as described plate was unaffected by the inclusion of up to 0.6% Sarkosyl under “Experimental Procedures.” during the elongation phase of the reaction (data not shown). The Effect of ~ u c ~ oConcentrations t ~ e on Initiation-In a transcription in this system remains committed to the major system reconstituted with fractions, we had found that ATP and CTP must be added to preformed rapid start complexes late promoter for more than one round of initiation events. in order to pass the stepblocked by 0.05% Sarkosyl(1). Other combinations of nucleotides, including ATP and UTP or any EXPERIMENTALPROCEDURESANDRESULTS’ nucleotide added singly to the reaction, failed to result in Sarkosyl Titrations-The concentrations of Sarkosyl re- Sarkosyl resistance, The simplest explanation for this obserquired to inhibit transcription initiation from the major late vation was that Sarkosyl resistance required minimal~y the promoter in a nuclear extract system were determined in the formation of the first phosphodiester bond, since the major experiment ofFig. 2. The protocol followed is summarized late RNA begins pppACUCU. . . . schematically below the figure. The DNA wasincubated with The nuclear extract contains small amounts of nucleotides the nuclear extract for60 min a t 30 “C. Sarkosyl, at the and, therefore, there was a possibility that initiation of tranconcentrations shown in thefigure, was present eitherduring scription could occurwithout added nucleotides. However, the the entire incubation (curve a) or only during the last 30 s of experiment of Fig. 2 showed that addition of nucleotides was the incubation (curve b). Nucleoside triphosphates were then required to confer resistance to 0.1% Sarkosyl, indicating that added, followed 30 s later by addition of Sarkosyl to 0.08%. the concentrations of endogenous nucleoside triphosphates The reaction was stopped 45 min after nucleotide addition were not sufficient to support productive initiation. This result led us to examine the nucleotide concentration require’ Portions of this paper (including “Experimental Procedures,” part ments for initiation as well as the rate of degradation of of “Results,”Figs. 1 and 3, and Table I) are presented in miniprint nucleotides in our extracts to determine whether rapid deplea t the end of this paper. The abbreviations used are: kbp, kilobase pair; nt, nucleotide;Hepes, 4-(2-hydroxyethyl)-l-piperazineethane- tion of nucleotides could leadto a decreasing rate of transcripsulfonic acid. M~niprintis easily read with the aid of a standard tion initiation. magnifying glass. Full size photocopies are available from theJournal In the experiment of Fig. 3, which is described in detail in of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. t we found that all four nucleoside Request Document No. 86 “3237, cite the authors, and include a the ~ i n i p r i n Supplement, (kA 20 min) when triphosphates were rapidly hydrolyzed check or moneyorderfor$3.20per set of photocopies. Full size photocopies are also includedin the microfilm edition of the Journal added to the nuclear extract at concentrations standard for that is available from Waveriy Press. the in vitro transcription assays. The addition of phosphoI

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Steps in Initiation Reinitiation and

3454

creatine to the reaction decreased the rate of hydrolysis by about 5-fold. The practical importance of these observations relates to the possibility that the degradation of nucleotides can affect the rateof transcription in a standardassay. In the experiment of Fig. 4, we tested directly whether nucleotide degradation would limit the ability of a transcription system to support more than one round of RNA chain initiation. After the major late promoterhad been incubated with nuclear extract for 50 min at 30 "C, we added nucleoside triphosphates. At the indicated times following this addition, we removed and quenched samples of the reaction for analysis of the number of full-length transcripts thathad been completed. We compared the patternof RNA synthesis infour reactions. Phosphocreatine was present in two reactions (open symbols), one of which also contained 0.025% Sarkosyl to limit transcription to one round of initiations (open circles). Phosphocreatine was omitted inthe othertwo reactions (solid symbols); again, 0.025% Sarkosyl was added to one of these reactions (solid circles), so that the level of transcript produced in one round of initiations could be measured. The results of this experiment demonstrated that in the presence of phosphocreatine, the amount of transcript observed in the absence of Sarkosyl continued to increase beyond the amountcorresponding to one round of transcription. However, whenphosphocreatine was not included in the reaction, the amount of transcript reached a plateau equivalent to a single round of initiations, even in the absence of Sarkosyl. Phosphocreatine did not affect the amount of transcript observed when transcription was limited to one round. In addition, neither Sarkosyl nor phosphocreatine had asignificant effect on the rate with which the transcripts were elongated in the first round of transcription. These results are consistent with the hypothesis that a deleterious effect of nucleotide degradation on initiation is manifested earlier than a possible decrease in the

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FIG. 4. Effect of nucleotide degradationon the reinitiation of transcription ina nuclear extract. RsaI cut pMLH2 DNA was incubated with nuclear extract for 50 min a t 30 "C in 80 pl, using the standard conditions described under "Experimental Procedures." Nucleotides with (0,A) or without (0,A) 10 mM phosphocreatine were added. To two reactions (0,O ) ,Sarkosyl was added in 5 pl to a final concentration of 0.022% 30s before nucleotide addition; 5 pl of water was added to the other two reactions (A, A). At the times shown in the Figure, 18-pl samples were removed and added to stop mixture; in addition, portions of each reaction were removed at 10,20,40,and 60 min and the fraction of label remaining as GTP was determined as described under "Experimental Procedures" and in the legend to Fig. 3. In this experiment, one round of initiations resulted in 73 pM transcript, which corresponded to 3300 cpm incorporated into the transcript.

by R N A Polymerase 11 TABLE I1 Nucleotides required for productive initiation from the major late promoter in the nuclear extract system Nucleotides at the concentrations shown were incubated with pMLH2 DNA and nuclear extract for 50 min at 30 'C in the absence (-PC) or presence (+PC) of phosphocreatine. The DNA concentration was 35 pg/ml;other solution conditions were as described under "Experimental Procedures." Sarkosyl was added to 0.1%, followed 30 s later by addition of all four nucleoside triphosphates, including [m3'P]GTP. Incubation at 30"C was continued for 30 min, after which the reactions were quenched and processed as usual. The amount of labeled GTP incorporated into transcript was normalized to thatobserved in the reaction in which 0.6 mM ATP and CTP were present in the initial incubation in the absence of phosphocreatine. This value was 1500 cpm, which corresponded to 2.0 fmol of 306nucleotide transcript in a 25-pl reaction. Nucleotides

% initiated complexes

-PC

+PC

-NTPs I% was visible as a distinct band on the autoradiogram. Productive initiation did not occur when nucleotides were omitted, even in the presence of phosphocreatine. However, the addition of ATP, CTP, or UTP alone produced some stably initiatedcomplexes whenphosphocreatine was also present during the incubation. The ability of 0.6 mM CTP to produce the greatest number of initiated complexes was consistent with an independent determination of the concentrations of ATP and CTP required for productive initiation from the major late promoter in the fractionated system, which indicated that the apparent K, for CTP was about 10-fold higher than that for ATP.' The fact that each

* D. K. Hawley, unpublished observations.

Steps in ~ ~ i t i u t i and o n ~ e ~ n ibyt RNA ~ uPolymerase ~ ~ ~ II of the nucleotides was able to promote productive initiation in the presence of phosphocreatine indicated that the other nucleotides were present in the extract, presumably in the form of di- andfor monophosphates. (The preparations of nucleotides used were high performance liquid chromatography purified and were shown not to contain contaminating nucleoside triphosphates by their inability to promote productive initiation in any but the expected combinations in the fractionated system. However, we cannot rule out the possibility that thenucleotides contained contaminating amounts of the nucleoside di- and monophosphates. This possibility does not alter the main conclusion of this experiment, however.) In thepresence of phosphocreatine, 0.6 mM ATP and CTP appeared to result only in a low level of initiation. This result was misleading, because the low incorporation was actually the result of extensive elongation before addition of the labeled GTP to thereaction. We determined this fact directly by monitoring the decrease in label incorporated into the transcript with increasing time of incubation of rapid start complexes with ATP and CTP before addition of Sarkosyl and labeled nucleotides (data not shown). Extent of Rapid Start Complex Formation not Limited by Availability of Transcription Factors-The time required to form rapid start complexes on the major late promoter was measured by adding promoter-containing DNA to nuclear extractand, at subsequent times, adding samples of this reaction to nucleotides and 0.03% Sarkosyl. The amount of transcript produced after an additional 30-min incubation was then determined and is shown in Fig. 5. The formation of rapid start complexes proceeded with apparent first order kinetics with a tlh of 8 min. The reaction was apparently complete after 50-60 min; further incubation before nucleoI.OC

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Extract hrbGiP’S FIG.5. Time required for formation of rapid start complexes on the major late promoter. To initiate the reactions shown, pMLH2 DNA was added to the nuclear extract in a volume so that of 220 pl.The DNA and extractwere preincubated separately both were at 30 “C when the reaction was initiated;thesolution conditions were as described under “Experimental Procedures.” At the indicated times, 2 0 4 portions were removed and addedto 0.04% Sarkosyl; 30 s later,nucleosidetriphosphates wereadded and the reactions were incubated an additional 30 min at 30 “C. The final concentration of Sarkosyl was 0.028%in a final reaction volume of 27 pl. The fractional amountof transcript producedin each reaction (F) wasdetermined by normalizing to the maximumamount of transcript observed (128 PM). The single exponential curvedrawn through the points was fit to the early (F< 0.8) time-points;tsn= 8 min.

3455

tide addition did not increase the number of complexes (data not shown). In thecourse of these studies, we used three different major late promoter-containing plasmids (pMLH1, pMLH2, and pSmaf; see “Experimental Procedures”) and found that although the rateof rapid start complex formation did not vary significantly among the plasmids, the extent of the reaction did. When we vaned the DNA concentration and measured the amount of transcript produced in thepresence of 0.025% Sarkosyl following a 60-min incubation of DNA with nuclear extract, we found that transcription increased with DNA concentratjon until a concentration of 35 pg/ml was used.At that and higher concentrations (up to at least 60 pg/ml), the amount of transcript remained constant. The lowest DNA concentration required for maximum transcription (35 pg/ ml) was the same for all of the plasmids; however, the maximum level of transcription was approximately 2-fold higher for pMLHl and pMLH2 than for the larger plasmid pSmaf. It is important to note that the amount of transcription approached at most one or two transcripts produced for every 100 promoters added to the reaction, so that the number of promotersper sedid not limitthe maximum level of transcription. One possible explanation for the similar dependence on total DNA concentration but different final level oftranscription among the different plasmids was that the extent of rapid start complex formation was not limited by the quantitative binding of a limiting transcription factor but, rather, was determined by the outcome of a competition between complex formation and inactivation of the DNA template. We therefore wanted to determine whether the components of the reaction that were apparently “depleted” during complex formation were the transcription factors or the DNA. We incubated a plasmid containing the major late promoter (pMLH2) with nuclear extract for 60 min; we then added a second major late promoter-containing plasmid (pMLH1) which produces a run-off transcript of a different size (diagrammed in Fig. 1). At various times following addition of the second template, we removed samples of the reaction and added 0.025% Sarkosyl and nucleotides, and allowed the preformed rapid start complexes to produce transcripts. The outcome, shown in Fig. 6, was that even though rapid start complex formation had gone to completion on the template present in the first 60min incubation (open circles), rapid start complexes couldstill form on thetemplate added subsequent to this firstincubation (solid circles).Furthermore, these complexes did not form on the second template by depleting complexes from the first template, because the number of rapid start complexes committed to thefirst templatedid not decrease during the second incubation period. The results of this experiment conclusively demonstrated that the extent of rapid start complex formation was not limited by the availability of transcription factors. Since a second DNA was able to form rapid start complexes, some acquired property of the first DNA must have been responsible for limiting the reaction to the observed level. The experiment of Fig. 6 also demonstrated the stability of the rapid start complexes to 0.025% Sarkosyl, even in the presence of additional template. When 0.025% Sarkosyl was included in the second incubation, there was no loss of rapid start Complexes from the first DNA during the second SO-min incubation in either the absence (open squares) or presence (open t r ~ ~ l eofs the ) second major late promoter-conta~ning template. As expected, no rapid start complexes formed on the second template in the presence of Sarkosyl (solid triangles). The long lifetime of rapid start complexes was also

Steps in Initiationand Reinitiation by RNA Polymerase I1

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FIG. 7. Transcription remains committed to a major late template for more than one round of transcription. RsaI cut stop pMLH2 DNA was incubated with nuclearextract at 30 "C for 66 min EXIFOCt in a volume of 725 pl. The DNA concentration was 30 pg/ml; the FIG.6. Transcription from a second major late template solution conditions were as described under "Experimental Proceafter completion of rapid start complex formation on a first dures." 110 & of the incubated reaction were then added to 4 pl of major late template. RsaI cut pMLH2 DNA (30 pg/ml) was incu- Sarkosyl(0,O) or water (m, U) and 6 pl of DNA or buffer as follows: bated with nuclearextract for 60min at 30 "C in a volume of 475 pl. panel A, 10 mM Tris, pH 8 , l mM EDTA; panel B, SmaI cut pMLHl This mixture was then split into three reactions, with 137.5 pl of the DNA; panel C, pUCl3 DNA. Nucleotides in20 pl were addedto each incubated mixture increased to 150 p1 by addition of Sarkosyl to reaction 30s after theee additions. The final concentrations 240 in pl 0.025% (a),4 pg of SmaI cut pMLHl DNA (0,O),or both the secondwere: Sarkosyl, 0.025%; pMLH2 DNA, 23 Clg/ml; pMLH1 or pUC13 DNA template and Sarkosyl(A, A). A t the indicated times following DNA, 23 pg/ml. A t the times indicated in the Figure, 17 pl of each these additions, 19-pl portions of each reaction were removed and reaction were added to 20 p1 of stop mixture and the reactions were added to the four nucleoside triphosphates and the amount of Sar- processed and electrophoresed on gels as described under "Experikosyl required to bring the final concentration to 0.025% in a total mental Procedures." The amount of 3~-nucieotidetranscript (exvolume of 24 pl. These transcription reactionswere incubated for 45 prewed as fmol transcript) from the pMLH2 template (solid symbols) min at 30 "C. The quenched reactions were processed and analyzed and 536-nucleotidetranscript from the pMLHl template(open symby ~ l y a c ~ l a m i dgel e electrophoresis as described under "Experib o l s ) was determined and is plotted as a function of time after the mental Procedures."The amountsof the run-off transcripts from the addition of nucleotides. pMLH2template (open symbols) and from thepMLHltemplate (solid symbols) were determined and plotted versus the elapsed time ~ indicates t ~ the mean value multiple rounds from reactions to which no second DNA was of the second incubation.The ~ r i z oline fortheamountof transcript produced from the pMLH2 (DNA1) added (panel A ) , to which a second DNA containing the major pM). The line fitting the values late promoter was added (panel B ) , and towhich vector DNA template in all the reactions (43 obtained for the pMLH1 template (DNA21 in the reaction to which c o n ~ i n i n gno bona fide class I1 promoter was added (panel Sarkosyl was absent in the second incubation (0)is a single expo- Cf. nential curve approaching a maximum value of 18.6 pM with tw = 9.9 Comparison of the level of transcription observed in the min. presence and absence of Sarkosyl showed that reinitiation did observed previously in the transcription system reconstituted occur in allof the reactions to which Sarkosyl was not added. Although neither the vector nor the second major Iate prowith fractions. Transcription Rernuins Committed to a Template for More moter-containing DNA significantly affected the amount of than One Round of ~ ~ ~ ~ ~ t ~ o ~ - S aconcentrations rkosyl of the transcript observed in one round of synthesis, both reof transcript observed 0.015% and 0.025% in the t~nscription systems reconstituted duced, to thesame extent, the amount withfractions and nuclear extract, respectively, not only in reactions in which transcription proceeded beyond one prevented formation of rapid start complexes, but also pre- round of in~tiation.(Compare soEid squares in Fig. 7, panel A, vented reinitiation of transcription, demonstrating that the with those in panels I? and C.) We concluded, therefore, that Sarkosyl-sensitive step must be repeated with each initiation this reduction was neitherdependent on nor significantly event. However, it was not known whether all of the steps affected by the presence of the major late promoter in the necessary for a first round of transcription must be repeated second DNA. In addition, following the completion of the with each subsequent initiation, or whether some transcrip- first round of transcription, the first template (panel B, solid tion factors remainassociated with thepromoter duringtran- squares) was transcribed approximately &fold better than the scription. An answer to thisquestion required the ability both second major late template (panel B, open squares), although to demonstrate that reinitiation had occurred and also to the concentrations of the two promoters were the same. (A low level of transcription of the second template was expected determine whether the reinitiations occurred on the same because, as described above, rapid start complexes could form templates used in the firstround of initiations. Our strategy for addressing this problem is shown sche- on a second template in the absence of transcription and matically in the lower part of Fig. 7. We allowed rapid start without loss of complexes from the first template underthese complex formation between the major later promoter and conditions, which were essentially the same as those of Fig. factors in the nuclear extract to go to comp~etion,We then 6.1 We have repeated these experiments with a higher concensplit thepreincubated mixture into six reactions. To three of these reactions, we added 0.025% Sarkosyl so that only one tration of DNA in the first incub~tion in an attempt to round of transcription could occur. The other threereactions sequester the limiting transcription components before the contained no Sarkosyl and transcription could reinitiate fol- addition of the second DNA. However, this approach had a lowing the first round of transcription. We then followed the technical limitation, in that high concentrations of template added subsequent to the first incubation depressed the level amount of transcript produced in a single round and in 1 DNA1

0' I

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Initiation Steps in

and Reinitiation by RNA Polymerase I1

of transcription to such an extent that it was not always possible to be certain that reinitiation had occurred. We have also tried reducing the amountof extract, insteadof increasing the amount of template, but have not succeeded in finding conditions that will completely exclude formation of rapid start complexes on the second template, when assayed according to theprotocol of Fig. 6. We therefore cannot rule out the possibility that some of the transcription of the second DNA was dependent on factors lost from the first template. However, as we have never seen an equal amount of transcription from the first and second added templates after the first round of transcription, we favor the conclusion that some transcription factor(s) tended to remain committed to the template at least through two rounds of transcription. Kinetics of ~einit~ation-In experiments where the accumulation of transcripts after additionof nucleotides to rapid start complexes was monitored (e.g. Figs. 4 and 6),we have consistently observed that after completion of one round of synthesis, continued synthesisproceeded apparently linearly but ata slower rate than thatobserved initially. One possible interpretation of this observation is that fewer rapid start complexes participated in the second round of synthesis than in the first round. A second possibility is thatthetime required to reform rapid start complexes was significant relative to the time required to elongate the transcripts to full length. In the experiment ofFig. 7, when additional DNAwas added to thereaction at thesame time as nucleotides (panels B and C), the final rate of accumulation of transcript was the same as that observed when no additional DNA was added (panel A). However, the total amount of transcript produced in 75 min was lower in the reactions of panels B and C for one of two possible reasons. 1) The lower rate of synthesis was achieved earlier in the reaction (at about 5 min, as indicated by the extrapolated dotted lines in panels B and C), before the completion of one round of synthesis. 2) The reaction completed one round of synthesis normally but the entry of the reaction into the second round was delayed. Either interpretation is consistent with the experiment of Fig. 7, although the results of similar experiments (not shown) suggested that thefirst round of transcription was completed normally, while the length of the delay in beginning the second round was variable and was increased by increasing the final concentration of DNA added to thereaction. In order to understand boththe decreased rate of transcript accumulation beyond the level of one round and theeffect of additional DNA on the kinetics of multiple round transcription, we directly measured the rate at which new rapid start complexes formed after addition of nucleotides to preformed rapid start complexes. In the experiment of Fig. 8, rapid start complexes were formed on the major latepromoter in a nuclear extract. Nucleotides were then added and, at subsequent times, samples of the reaction were removed, added to 0.025% Sarkosyl, and thenincubated for an additional 50 min so that all of the rapid start complexes that had formed at the time of Sarkosyl addition could complete transcripts (indicated by circles). In a separate reaction, a second major late promoter-con~iningtemplate was added at the same time as nucleotides and theamount of transcript produced by the template added first (open squares)or second (solid squares) was determined. Vector DNA lacking the major late promoter was added to athird reaction (triangles). The amount of RNA produced from the rapid start complexes already formed by the time of nucleotide addition (indicated inthe Figure with an arrow) was determined by adding

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DNA I Extract

NTP'S Stop Sarkosyl -DNA2 0 +DNA2 0,. +pUC A FIG. 8. Time required for formation of new rapid start complexes after initiation oftranscription on the major late promoter. RsaI cut pMLH2DNA wasincubated with nuclear extract at 30 "C for 50 min using standard reaction conditions in a volume of 450 pl. The DNA concentration was 30 pg/ml; 110 pl of the incubated mixture was added to each of three 30-pl mixtures already a t 30 "C and containing nucleotides and phosphocreatine (to bring the final concentrations to standard conditions for these components) and either no additional DNA (O), SmaI cut pMLHlDNA (01, or pUC13 DNA (A). The final concentration of pMLH2 DNA was 24 pg/mI in all reactions. The final total DNA concentration was 48 pg/ml in reactions in which additional DNA was present. At the indicated times after addition of the preformed rapid start complexes to nucleotides, 25-p1 samples were removed and added to Sarkosyl (final concentration, 0.025%) to block further rapid start complex formation. These small reactions were incubated an additional 50 min after Sarkosyl addition to allow full-length transcript production from all rapid start complexes. The amount of 306-nucleotidetranscript from the pMLH2 template (open symbols) and 536-nucleotide transcript from the pMLHl template (B)was determined and is plotted as a function of time after addition of nucleotides. The arrow indicates the amount of transcript produced when Sarkosyl was added to 0.025% to a portion of the initial incubated reaction 30 s before nucleotides. This reaction contained the same components in the same concentrations as theother reactions and was treated identically in all respects other than order of addition of Sarkosyl and nucleotides.

Sarkosyl before nucleotides and incubating for 50 min as for the other reactions. This experiment showed that addition of nucleotides was followed bya lag phase (at least 5 min) in which no new rapid start complexes were formed. When a second promoter-containing DNA was added with nucleotides, the length of this lag was increased but the kinetics of the subsequent reaction were essentially identical to those whichfollowed the lag phase in the absence of a second DNA. As was also observed in the experiment of Fig. 7, the effect of the second DNA on the kinetics of the reaction was not dependent on the presence of the major late promoter, since the amount of transcript produced in the reactions to which pUC or pMLH1 DNA were added was similar at every time point. Although rapid start complexes did form onthepMLHl template (solid

3458

Initiation Steps in

and Re~nitiationby RNA Polymerase 11

squares), the number of these complexes was only about onefifth the number of new complexes formed on the pMLH2 template (DNA1) after nucleotide addition. This result provided additional support for the conclusion that the DNA template used in the firstround of RNA synthesis was transcribed in the second round in preference to newly added DNA. The kinetics of initial formation of rapid start complexes and the reformation of complexes following nucleotide addition were clearly different, because no lag phase was observed when nuclear extract and the template were first combined (Fig. 5 ) . These observations suggested that the transcripts initiated during the first round of synthesis must undergo some elongation before new rapid start complexes begin to form. This eiongation could be necessary either to free the promoter for the binding of new transcription factors or to release some component of the reaction that must finish elongation before becoming available for the formation of new rapid start complexes (e.g. the polymerase). This second possibility seemed less likely because of the observation that the formation of rapid start complexes was not limited initially by the availability of transcription factors (Fig. 6). We have done preliminary experiments to test the effect of the elongation rate on the rate at which reformation of rapid start complexes occurred. We found similar lag periods for a variety of elongation rates (adjusted by varying the UTPconcentration). For example, in one side by side comparison of two reactions, we found the same number ofnew rapid start complexes formed by 10 min after addition of nucleotides. A separate measurement of the elongation rates in these reactions showed that within 10 min all of the transcriptsinitiated in the first round had been fully elongated in one reaction, whereas less than 25% of the transcriptshad been completed in theother reaction.2 This result strongly suggested that the completion of transcripts was not required for formation of new rapid start complexes, but left open the possibility that promoter clearance was the rate-limiting event. Alternative possibilities for the lag include a rate-limiting step that was unique to theprocess of reinitiation or a substantial reduction in the effective concentration of a transcription factor due, for example, to nonspecific binding to DNA or to instability at 30 "C.

0.025 %

0.1 %

Sorkosyl I

Sorqosyl I

FIG.9. Proposed model for steps in transcription initiation by RNA polymerase I1 inanuclear extract. Two steps in transcription initiation in a nuclear extract can be separated by the use of different concentrations of Sarkosyl. Formation of the rapid start complex, which can initiate transcription within 30 s from the major late promoter, is inhibited by concentrations of Sarkosyl >0.025%. Conversion of the rapid start complex to a stably initiated complex is inhibited by concentrations of Sarkosyl >0.1%. At some point after initiation of transcription, transcription is preferentially reinitiated at the same promoters used for the first round of initiations. The step that is circumvented in the second round of transcription precedes the stepblocked by 0.025%Sarkosyl.

a step preceding formation of the rapid start complex, which was inhibited by0.025% Sarkosyl in the second round of initiation as well as thefirst. The issue of whether or not a template containing a class I1 promoter remains committed to transcription throughmultiple rounds of initiation events could not previously be resolved in uitro. In the RNA polymerase I and I11 systems, an unambiguous demonstration that transcription factors do remain bound to a promoter through multiple initiation events was possible because the total amount of transcription observed was greater than one transcript for every promoter present in the reaction, proving that reinitiation must have taken place; yet there was no loss of factors to competing templates (15-17). Incontrast, most RNA polymerase I1 transcription systems (at least the unfractionated extracts) produce at most about one transcript for every 10 promoters in astandard reaction (14, 18). Withouta way to block reinitiations, it was not possible to determine that they had occurred. (Reinitiation is defined here as an initiation event that could not take place until after a previous initiation event.) The experiments presented here and in our previous publication (1) demonstrated that reinitiations were occurring in our transcription systems and, furthermore, showed that the level of transcription without reinitiations could be deterDISCUSSION mined. We used this technique to show that when transcripThe experiments we have presented here showed that sev- tion wasallowed to continueafter preformed rapid start eral functional steps in transcription initiation could be ob- complexes had initiated, the template on which these rapid served in a nuclear extract system using appropriate concen- start complexes had been formed was preferred in the second trations of Sarkosyl. Two stepsapparently correspond to round of initiations over a second template added at thesame those observed previously with the more purified system, time as nucleotides. Because only a small percentage of the namely, formation of the rapid start complex and formation promoters present during the first incubation were actually of a productively initiated complex. Wehad previously defined transcribed, we could not show conclusively that the same an additional step preceding formation of the rapid start promoters were transcribed in the first and second round of complex and resulting in a template-committed complex (1). initiations. However, that is the simplest explanation for the The ability to detect this complex relied on the finding that observation that thefirst templatewas preferred to thesecond for the following reason. A model that proposed use of differthe inhibition of rapid start complex formation by0.015% Sarkosyl was reversible when the Sarkosyl concentration was ent DNAlpromoters in preference to DNA2 promoters inthe decreased by dilution following the initial incubation. We second round of initiations would have to include an explahave not been able to repeat this result with the nuclear nation for why these DNAl promoters became activated only extract system, possibly because the higher concentration of after transcription from other DNAl promoters. We showed Sarkosyl needed to inhibit rapid start complex formation in the experiment ofFig. 6 that the extent of rapid start (0.025%) irreversibly inactivated some component necessary complex formation was not determined by the amount of a for transcription.' However, we have demonstrated a template limiting transcription factor. Therefore, the simple explanacommitted complex as an intermediate in the reinitiation of tion that release of transcription factors from elongation transcription in the nuclear extract system. As the model in complexes was necessary before reinitiation could occur is Fig. 9 illustrates, we propose that there is a continued asso- unlikely, although not ruled out completely. However, if the ciation of some component of the transcription machinery at release of a factor or factors were necessary for reinitiation,

Steps in Initiationand Reinitiation by RNA Polymerase I1 then we have narrowed the possibilities for when this release can occur. Weknow from experiments in the fractionated system that reinitiations from the major late promoter required the presence of all four nucleoside triphosphates. Omission of GTP, which resulted in elongation only to +lo, did not allow reinitiation (I).' On the otherhand, experiments in which the elongation rate was altered by changing the concentration of UTP showed that reinitiation did not depend on completion of transcripts. These considerations together suggest that the promoters themselves were the components that were reused during reinitiation. We do notyet know whether the factor(s) involved in committing the template to transcription in the second round are the same as those involved in the template commitment step defined in the earlier paper (1).Indeed, as the previous work useda transcription system reconstituted with fractions, it is possible that not all the factors involved in transcribing the major late promoter in the nuclear extract were present. For example, the partially purified system probably did not contain asignificant amount of the transcription factor USF, which has been shown recently by several groups to bind to a sequence upstream of the TATA box in the major late promoter and to increase the efficiency of transcription of this promoter in vitro (5-7). The majority of USF in the nuclear extract has been found to fractionate in the P11 0.3 M step fraction (5), whichwas not included in ourreconstituted system. We do not yet know whether USF is involved in the continued commitment of the major late promoterbeyond the first round of initiations nor is it possible from the data presented here to determine whether the step thatis circumvented in thesecond round of initiations was rate-limiting in the first round. These mechanistic details willbe pursued with further analysis. Our finding that transcription remained committed to a template containing the major late promoter for more than one round of initiations is in contradiction with the published results of Safer et al. (19)who failed to find such a preference for the first template in a transcriptionsystem reconstituted in vitro with wholecell extract.Apart from the obvious possible differences between the whole cell and nuclear extracts, another potential explanation for the difference between their results and those reported here is the number of rounds of transcription initiation that were observed in each case. In our experiments, the equivalent of only two rounds of transcription were completed. In the reactions of Safer et al. (19) the implication was that more than two rounds of transcription were observed, because they reported equal transcription from the template present during the initial incubation and the template added subsequently. In two rounds of transcription, one would expect to observe a greater level of transcript from the first template, to which the first round would be committed. Because Safer et al. (19) did not report either the actual number of transcripts produced in the multiple-round experiment or the number of transcripts expected in one round of initiations, it is not possible to resolve unambiguously the discrepancy between the two observations. Our model for the events occurring during reinitiation of transcription from the major late promoterin vitro is consistent with the recent observation in vivo of Mattaj et al. (20) who found that the promoter for the Xenopus U2 gene was transcribed for multiple rounds when injected into Xenopus oocytes, whereas a second template containing the U2 promoter, when injected after the first template, was not transcribed. The structureof the promoters for the small nuclear RNA genes does not bear an obvious resemblance to that of other class I1 promoters, including the major late promoter

3459

(21). However, it is interesting to speculate that the stable sequestration of some transcription factors plays a role in regulation of class II genes. The implications of such a mechanism for gene regulation have been discussed by others (22). Properties of the Nuclear Extract System-This study revealed three properties of the nuclear extract system that have important implications both in suggesting better ways to design reaction protocols and in advising caution in the interpretation of results obtained with this system. First, we found that nucleoside triphosphates werevery rapidly degraded in the nuclear extract and, more important, that this degradation had a marked effect on the ability of transcription complexes at themajor late promoter to initiate. This result suggested that an assay that measured the amount of transcript produced in one round of initiations from preformed rapid start complexes would be morereproducible and possibly also more efficient than an assay that measured transcript accumulation in a 30- or 60-min incubation at 30 "C following the initial mixing of all required transcription components. Both of these predictions were borne out by the experiment of Table I. A second property of the nuclear extract system that has practical importance is related to thenucleotide requirements for productive initiation. We showed previouslythat addition of both ATP and CTPwas required for productive initiation from the major late promoter in a fractionated system free of endogenous nucleoside triphosphates. In contrast, we found that with the nuclear extract system, addition of any single nucleoside triphosphate to a concentration of 600 PM was sufficient to allow some productive initiation to occur if phosphocreatine was also present. A lower concentration of ATP (100 PM) also promoted a detectable level of productively initiated transcription in the presence of phosph~reatine.It is likely that this result reflected the presence of pools of nucleoside diphosphates and/or monophosphates as we11 as the enzymes required to transfer phosphate groups, and that addition of any nucleoside triphosphate to the extract resulted in an increased concentration of all four nucleoside triphosphates. A similar phenomenon has been described previously for other cell extracts (23-25). The primary importance of this observation is related to thein~rpretationof experiments in which ATP was included during an initial incubation of template and extract inthe absence of the other nucleotides. From one such experiment, the conclusion wasmade that ATP was required prior to theformation of the first phosphodiester bond (26); however, there was no rigorous demonstration that some initiation had not occurred during the initial incubation. The third finding of practical significance was that the extent of formation of rapid start complexes was not limited by the availability of transcription factors. Therefore, the number of active transcription complexes formed in these extracts cannot be used reliably as a basis for calculations of the amounts of transcription factors in extracts or comparisons of the amountof factors in different types of extracts. REFERENCES 1. Hawley, D. K., and Roeder, R. G. (1985) J. Bwl. Chem. 260, 8163-8172 2. Matsui, T.,Segall, J., Weil, P. A., and Roeder, R. G . (1980) J. Biol. Chem. 265,11992-11996 3. Samuels, M.,Fire, A., and Sharp, P. A. (1982) J. Biol. Chem. 257,14419-14427 4. Dignam, J. D., Martin, P. L., Shastry, 3. S., and Roeder, R. G. (1983) Methods Enzymol. 101,582-598 5. Sawadogo, M., and Roeder, R. 6.(1985) CeU 43, 165-175 6. Carthew, R. W., Chodosh, L. A., and Sharp, P. A. (1985) Cell 43, 439-448

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Steps in ~ n i t ~ ~and i o Re~n~tiatwn n by RNA Polymerase II

7. M i y a m o ~ N. , G., Moncollin, V., Egly, J. M., and Chambon, P. (1985) EMBO J. 4,3563-3570 8. Dynan, W. S., and Tjian, R. (1983) Cell 32,669-680 9. Parker, C. S., and Topol, J. (1984) Cell 37,273-283 10. Jones, K. R., Yamamoto, K., and Tjian, R. (1985) Cell 42, 559572 If. Graves, B. J., Johnson, P. F., and McKnight, S. L. (1986) Cell 44,565-576 12. Cohen, R. B., Sheffery, M., and Kim, C. G. (1986) Mol. Cell Biol. 6,821-832 13. Weit, P. A., Luse, D. S., Segali, J., and Roeder, R. G. (1979) CeEl 18,469-484 14. Dignam, J. D., Lebovitz, R. M., and Roeder, R. G . (1983) Nucleic Acids Res. 1 1 , 1475-1489 15. Wandelt, C., and Grummt, I. (1983) Nucleic Acids Res. 11,37953809 16. Cizewski, V.,and Soliner-Webb, B. (1983) Nucleic Acids Res. 11, 7043-7056

17. Bogenhagen, D. F., Wormington, W. M., and Brown, D. D.(1982) Cell 28,413-421 18. Manley, J. L., Fire, A., Cano, A., Sharp, P. A., and Gefter, M. L. (1980) Proc. Natl. Acad. Sci. U. S. A. 77,3855-3859 19. Safer, B., Yang, L., Tolunay, H. E., and Anderson, W. F. (1985) Proc. Nut1 Acad. Sci. U. S. A . 82,2632-2636 20. Mattaj, I. W., Lienhard, S., Jiricny, J., and DeRobertis, E. M. (1985) Nature 316,163-167 21. Mattaj, I. W. (1984) Trends Biochem. Sei. 9,435-437 22. Brown, D. D. (1984) Cell 37,359-365 23. Saiga, H., and Higashinakagawa,T. (1979) Nucleic Acids Res. 6, 1929-1940 24. Korn, L. J., Birkenmeier, E. H., and Brown, D. D. (1979) Nucleic Acids Res. 7,947-958 25. Wilkinson, J. A. K., Miller, K. G., and Sollner-Webb, B. (1983) J. Biol. Chem. 258,13919-13928 26. Tolunay, H.E., Yang, L., Anderson, W. F., and Safer, B. (1984) Proc. Natl. Acad. Sci. U. S. A. 81,5916-5920

Supplementsly Materlal to Functional Steps In Transorlption InltlatlOn and Rslnltlatlan Prom the Major late Promoter ln B HeLa Nuclear Extraot

Dlane X, naxley and Robert 0. RoedeP Experimental Procedures

Pig. 1. The structures of t w o ineJor late promoter-contslnlna plasmids. pMLHl contalnaadenovlrus-2 sequenoes (Indicated by Bolld bBr8) extending from -260 to +536 with IIeSpeOt to the cap slte (+1) of the major late pP080ter. The pUCl3 Yeator sequences are repvesented with a dashed line. With SmaIl-estl.loted pKLHlas B tamplate, transcclption inltlated at the naJor m e PPomOteP PeRUltS in a 536-nt Pun-off' tianBePipt (lndlcated by an BLIrow below A transorlptianal a w e s i r e the disgran of t h e plasmld). in the adenovirus DNA. We (repressnteil by zigzags) 1s present at 8Pound *1[6 showd pmvlouslythat Oertaln Concentratlona of SaLlkosyl lnduced permanent phualng O f tFanSClliPtlon at this site. The pla9mld pMLH2 does not Oontsln thla pBuse site, because the adenovirus sequences extend only to 147. Restriction of pMLH2 at an RBBI Slte wlthin the Vector sequences Pesulted 1" B template that p~oducesB 3 0 G t tranacrlpt

-

-.

0.I

20

40

60

TIME Imin)

phosphocreatine, reapeotively.

In the experlment of Fig. 3, o~eatlnephospholtlnase YBB not added to the reaction contalnlng PhOaphOCFeBtlne. demanstratlng that tha ensYmC * B E already present in the extract. In fact, addltlon of OPeatlne phoaphoklnase had no deteotable effect on the rate of deCay (data not shown). Creatlne phosphokinase dlreCtly phoaphorylates ADP; in Order for the Other nucleotldea to be protected from degradation b$ phosphocreatine. the extrsct !WSt Contain the Xlnases responalble for transferring phosphate groups from ATP to the OtheP nueleoslde mono- and diphosphates. This " 8 8 apparently true f o r all of the extracts we tested; addition af nUOleoslde dlphosphoklnaae to the eXtPaOt had no effect on the rate of degradatlor. of GTP (data not shorn). We ala0

3461

Steps in Initiation and Reinitiation by RNA Polymerase 11

Extraot +

Sarkosyl PC

+

PC

after 60 minPC +PC

1

1.811

1 .00

2.77

2

1.81

0.68 21.5

0.74

3

1.98

1.85

2.76

4

1.59

1.31

0 .89.0 96

20.3

0.47 5

1.59

1.39 91.U

27.8

5 Mean 2 8.d. 1.76

1.43 1.75

1.31

z .15

1.10 2 .50

13.5

85.8

N.D.

N.D.

90.6

37.5 1.67 2 .89

92.2