Separation and Partial Characterization of Three Functional Steps in ...

Vol. 260, No. 13,Issue of July 5, pp. 8163-8172, 1985 Printed in U.S. A.

THEJOURNAL

OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc

Separation and Partial Characterization of Three Functional Steps in Transcription Initiation byHuman RNA Polymerase 11* (Received for publication, January 28,1985)

Diane K. HawleyS and Robert G. Roederfj From The Rockefeller University, Laboratoryof Biochemistry and Molecular Biology, New York, New York 10021

Unlike the purified E. coli RNA polymerase, purified RNA polymerase I1 cannot begin specific transcription from promoters i n vitro but must be supplemented by other proteins present in crude extracts (5). Chromatographic fractionation procedures have separated HeLa extracts active for specific RNA polymerase I1 transcription into at least five fractions (called TFIIA, -B, -C, -D, and -E1) required to reconstitute a system that accurately transcribes theadenovirus major late promoter (6-8). The active component in one required fraction, TFIIC, was identified as poly(ADP-ribose) polymerase and shown tosuppressrandomtranscription in vitro by binding to nicks in the template (9); it can be omittedwhen the other factors are more highly purified (9). The role of other putative transcription factors is not known, although there is both direct and indirect evidence that several of these factors interact with particular DNA sequences within the promoter (10-13).’Because the standard protocols for assaying specifictranscription require production of a discrete runoff transcript or a transcript that can be analyzed with S1 nuclease, it has not yet been possible to determine whether the various transcription factors act exclusively during initiation or elongation. Regulation of the frequency of transcription initiation is an As a first step toward understanding the component steps important means of controlling gene expression in bacteria, of RNA polymerase I1 transcription and the factor requireand there is accumulating evidence that this is also true in ments for these steps, we sought to separate initiation from higher organisms. Much progress has been made recently in subsequent elongation events by limiting transcription to a characterizing the rate-limiting steps in transcription initia- single round of initiations. We focused on the anionic detertion inEscherichia coli and correlating initiationfrequency i n gent Sarkosyl as a means of preventing reinitiations in vitro because Sarkosyl has been known for a number of years to vivo with the measured rate of open complex formation i n vitro (1).Furthermore, studies measuring the qualitative and inhibit initiation but not elongation by eucaryotic RNA polymerase I1 (14). Furthermore, at Sarkosylconcentrations quantitative effects of several regulatory proteins on the separound 0.5%, alarge fraction of the chromatin-associated arated steps in initiation have demonstrated that both the proteins is solubilized (15, 16), resulting in a stimulation of initial binding of RNA polymerase to the promoter and the RNA polymerase I1 transcription in isolated chromatin (17) subsequent conversion of this complex to a transcriptionally and nuclei (15, 18,19). These resultssuggested that Sarkosyl active form are targets of positive regulation in E. coli (2-4). would be an effective reagent even in the crudest reconstituted One of the goals of this and other laboratoriesis to obtain systems. an understandingof transcription by eucaryotic RNA polymIn this paper we describe an analysis of RNA polymerase as is now available for erase I1 in the same mechanistic detail I1 transcriptioninitiation ina systemreconstituted from procaryotic transcription.However, progress in thisfield has partially fractionated HeLa nuclear extracts. We found not been slow, partly because of the absence of both genetic and only that Sarkosyl effective is in blockingreinitiations in vitro i n vivo data needed to corroboratei n vitro results and partly but also, more importantly, that we could separate initiation because of the necessity of studyingRNA polymerase I1 of transcription into three steps on the basis of differences in transcription in impure and relatively complex i n vitro sys- sensitivity to Sarkosyl. These results are a very promising tems. indication thatfairly detailed mechanistic information can be *This work was supported by Grants CA 34223 and CA 34891 obtained even from cruderelatively unfractionated systems. from the National Institutes of Health t o R. G. R. and by Grant CD EXPERIMENTAL PROCEDURES 203C from the American Cancer Society. The costs of publication of Materials and DNA Template-Sarkosyl (N-lauroyl sarcosine sothis article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in dium salt) was purchased from Sigma. Unlabeled nucleotidespurified accordance with 18 U.S.C. Section 1734 solely to indicate this fact. * The abbreviations used are: TF, transcription factor; Hepes, 43 Supported by a postdoctoral fellowship from the National Insti(2-hydroxyethyl)-l-piperazineethanesulfonic acid. tutes of Health. N. Nakajima and R. G. Roeder, manuscript in preparation. I To whom correspondence should be addressed.

We have used Sarkosyl to study the events comprising specific transcription initiation in vitro by HeLa RNA polymerase 11. On the basis of different sensitivities to the Sarkosyl concentration, we have defined three functional steps in initiation at the adenovirus major late promoter: 1. a template commitment step that occurs in the presence of 0.015% Sarkosyl; 2. a nucleotide-independent conversion of the committed complex to a “rapidstart complex”capable of initiating an RNA chain, a step blocked by 0.015%Sarkosyl; and 3. a step that requires nucleoside triphosphates, converts the rapid start complex to a stably initiated complex, and is sensitive to Sarkosyl concentrations >0.05%. The subsequent elongation of the initiated RNA chain is resistant to Sarkosyl, except that Sarkosyl causes pausing or premature termination at a specific site about 186 nucleotides downstream of the major late cap site. Using this assay, we have further characterized these steps and the resulting intermediate complexes.

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Steps in RNA Polymerase 11 Transcription Initiation

by high-performance liquid chromatography were purchased from ICN. [N-~'P]GTPwas from New England Nuclear. The DNA used in the transcription experiments was the plasmid pSmaf, which contains the 2.4-kilobase SmaI F fragment of adenovirus 2 ligated into the SmaI site of pBR313(5).Thisplasmidcontainsthe major late promoter and 536base pairs of downstream sequences. Fractionation of Nuclear Extracts-HeLa cell nuclear extractswere prepared and subjected to chromatographic fractionation on phosphocellulose (Whatman P-11) as previously described (6),except that all buffers contained 20 mM Tris, p H 7.9, at 4 "C instead of Hepes. Proteins eluted in three of the four KC1 step fractions from P-11 chromatography are required vitro in to reconstitute RNA polymerase I1 specific transcription: the breakthrough (0.1 M KC1); the 0.5 M KCI; and the 1 M KC1 fractions (7). TheP-11 0.5 M KC1 fraction was dialyzed against BClOO (20 mM Tris, pH 7.9 at 4 "C, 20% glycerol, 0.1 M KC1, 10 mM 2-mercaptoethanol, and 0.2 mM EDTA) and was not processed further. This fraction (about 2.4 mg of protein/ml) contained TFIIB, TFIIE, and RNA polymerase I1 (6, 7). The activity in the P-11 1 M KC1 fraction was concentrated about 2- to 3-fold by precipitationwith 60% ammonium sulfate. The pellet was resuspended to a concentration of2.5 mg of protein/ml in BCO (same composition as BClOO but without KCI) and 0.1% Tween 40 and was dialyzed against this same buffer to a final salt concentration of 0.1 M KC1. (In some experiments, a preparation wasused that was dialyzed against BClOO without detergent. There was no significant difference in the activity or stability of the two preparations.) We found that the P-11 1 M KC1 fraction, which contains TFIID and poly(ADP-ribose) polymerase (also called TFIIC), also apparently contains inhibitors of the transcription reaction. Thus, a titration profile for this fraction did not reach a plateau but showed a sharp peak of maximum transcription activitya t around 250 pg/ml protein. The ammonium sulfate precipitation step,while increasing the maximum amount of transcription observed, did not appear toaffect the pattern of titration significantly. The P-11 breakthrough (0.1 M KCI) fraction contains the required factor TFIIA. Because this fraction also contains most of the nucleotides present in the nuclear extract, we used TFIIA that had been purified by two additional chromatographic steps. The protein from the P-11 breakthrough fraction (59 mg) was precipitated with 60% ammonium sulfate, dissolved in 1.0 ml ofBClOOO (which contains 1 M KCI), and chromatographed on a 96-ml Bio-Gel A-1.5m column equilibrated with BC1000. The column fractions containing TFIIA activity were pooled(3.7mg of protein in 5 ml), dialyzed against BC100, and loaded on a DEAE-cellulose (Whatman DE52) column equilibrated with BC100. After the column was washed with BC100, TFIIAactivity was stepelutedwith BC250 (0.25 M KCI). This fraction, containing 1.5 mg of protein in 7 ml, was dialyzed against BClOO and was stored in this buffer. Transcription Reactions-The protocol used for all transcription reactions includeda 30 "C preincubation of protein fractions and template DNA in the absenceof nucleotides, followed by addition of nucleotides and a second incubation a t 30 "C. The times of these incubations varied and are listed in the legends to the figures. The reaction volume was usually 20 p l during the preincubation, with nucleotides added in 5 pl. Alternatively, 20-pl samples were removed from larger preincubation reactions and added to 5-pl nucleotides. The solution conditions maintained in the first incubation were 12 mM Tris (pH 7.9 a t 4 "C), 12% glycerol, 60 mM KC1, 0.1 mM EDTA (all contributed by the storage buffer for the protein fractions), 20 mM Hepes, pH 8.4, a t 25 "C (to bring the final pH to 7.8 a t 30 "C), and 10 mM MgC1'. The template DNA,usually SmaI-digested psmaf, was added to a concentration of 20 pg/ml (approximately 2.5 nM promoter). The protein fractions were added to the following percentages of the final reaction volume: TFIIA, 12% (30 pglml), P-11 1 M KC1, 10% (240 pglml), P-11 0.5 M KCI, 18%(450 pg/ml). Nucleotides were added in water to final concentrations of 600 phi ATP, CTP, and UTP, and 15p~ GTP. [o~-~'P]GTP was added to a specific activity of 2-2.5 X lo5 cpm/pmol. Transcription reactions were stopped by adding an equal volume (usually 25 p l ) of transcription stop mix: 10 mM EDTA, 0.1 M sodium acetate, pH 5.5, 0.5% sodium dodecyl sulfate, 1 mg/ml yeast RNA. The quenched reactions were extracted once with an equal volume of phenol-chloroform; the organic phase was re-extracted with an equal volume of transcription stopmix. The aqueous phaseswere combined, and 2.5 volumes of ethanol were added to precipitatethe RNA. Following centrifugation, the RNA pelletswere redissolved in 15-20 p1 of 98% formamide plus dyes and electrophoresed ona 4.5% poly-

acrylamide (300.8 acry1amide:bisacrylamide)gel containing 7 M urea. The running buffer was 0.09 M Tris, 0.09 M borate, 2.5 mM EDTA. The gel was dried under vacuum and exposed to Kodak XAR-5 or BB-5 film. Qwntitation of Transcripts-Gel slices containing the transcript bands and slices of equal size from immediately above and below the transcript were cut from the driedgel with scissors. The radioactivity was measured by scintillation counting or, alternatively, Cerenkov radiation was measured. The cpm in the gel above and below the transcript were averaged and subtracted from the total cpm in the part of the gel containing the transcript to correct for background radioactivity. The two background measurements were usually within 5-10% of each other, while the average background determined in this manner contributed 20-30%of the total cpm in the specific transcript band. The picomolar amount of transcript was determined by measuring the total radioactivity added to each transcription reaction, calculating the fraction of total GTP incorporated into the transcript, and correcting for the number of guanosine residues in the transcript. In making this determination, we did not correct for the small amount of quenching of cpm by the polyacrylamide gel relative to free cpm in solution. Thus, the amounts of specific transcripts, while consistent within an experiment (because the quench factor was constant), were slightly underestimated. We also made no correction for the fraction of the added radioactivity that could actually be incorporated into RNA. This fraction, which is always less than 1.0, typically varies over a 2- to 3-fold range among different lots of [a-32P]nucleoside triphosphates, as was originally observed for [a-32P]UTP(20). RESULTS

Effect of Sarkosyl on Steps inInitiation-Our transcription system was reconstituted with transcription factors that had been partially purified from HeLa nuclear extracts in order to remove endogenous nucleotides (see "Experimental Procedures"). The DNA template was the plasmid pSmaf,which contains the major late promoter of adenovirus ( 5 ) . As outlined in the protocol in Fig. 1, we divided our transcription reactions into two steps: 1)an initial incubationof DNA and the protein fractions in the absence of nucleotides; and 2) a second incubation after addition of nucleotides to allow initiation and productionof a 536-nucleotide run-off transcript. We added Sarkosyl to the reaction at three different times: before addition of DNA to the protein fractions (point a), after the preincubation but 30 s before nucleotide addition (point b ) , and 30 s after nucleotide addition (point c). Using this protocol, we found that different concentrationsof Sarkosyl blocked transcription a t two differentsteps in the overall initiation process. As shown in the upper part of Fig. 1, a very low concentration of Sarkosyl(O.OO5X) had no effect on transcription relative to the control reaction without Sarkosyl (lane I ) . This result was observed when the Sarkosyl was included in the preincubation (lane 2 ) and when introduced immediately before or after the additionof nucleotides (lanes 3 and 4). A slightly higher concentration of Sarkosyl (0.015%) almost completely inhibitedtranscription when present during the preincubation(lane 5 ) but did not inhibit initiation of the RNA chain when added following the preincubation, as shown by the nearly identical amounts of transcription observed whenSarkosyl was added either 30 s before or 30 s after the addition of nucleotides (lanes 6 and 7). (We show below that under our standard conditions, transcription initiation iscomplete 30 s after nucleotide addition.) A higher concentration of Sarkosyl (0.25%) prevented initiation not only when present during the preincubation but also when added just prior to the nucleotides (lanes 8 and 9). When added after the nucleotides, this concentration of Sarkosyl greatly reduced the amount of the expected 536-nucleotide transcript andelicited, instead, a shorter transcript (lane 10). We haveused S1 mapping to show that this transcript is initiated at the major late promoter and to determine its

Steps inRNA Polymerase 11 Transcription Initiation I 2 3 4 5 6 7 8 9 1 0 c-

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DNA NTP'S stop Focrors FIG. 1 . Different concentrations of Sarkosyl inhibit different steps in RNA polymerase I1 initiation. Sarkosyl was added to individual transcriptionreactions a t threedifferenttimes, as indicated by the letters a, b, and c. In all cases, DNA and protein fractions in 20 pl were incubated at 30°C for 50min, using the standard conditions described under "Experimental Procedures." Nucleotides in 5 pl were then added, and the incubation at 30 "C was cont.inued an additional 40 min. Sarkosyl in 2 p1 was added to the reaction atthe following times: lanes 2, 5, 8, before the 50-min preincubation: lanes 3, 6, 9, after the preincubation but 30 s before nucleoside triphosphates (NTP:s);lanes 4 , 7, 10.30 s after nucleotides. The final Sarkosyl concentrations were: lane 1, no Sarkosyl; lanes 24, 0.005%; lanes -5-7, 0.015%; lanes 8-10, 0.25%. The DNA template (pSmaf) was cut with SrnaI so that arun-off transcript of 546 nucleotides was expected from the major late promoter. In the presence of 0.25% Sarkosyl, a shorter transcript (-186 nucleotides) was observed. This transcript, which originated from the major late promoter, is descrihed in more detail in the text and the legend to Fig. 2. The amount of specific transcript in each reaction was: lane 1,25.5 PM; lane 2. 27.1 PM; lane 3, 26.2 p ~ lane ; 4 , 26.2 PM; lanr 5, 0.71 p ~ lune 6, 14.6 PM: h e 7, 15.6 pM; h e 8, 0.02 pM; /On? 9,