MOLECULAR AND CELLULAR BIOLOGY, Nov. 1990, p. 5782-5795 Vol. 10, No. 11 0270-7306/90/115782-14$02.00/0 Copyright © 1990, American Society for Microbiology In Vitro Analysis of a Termination Site for RNA Polymerase II DEBRA K. WIEST' 2 AND DIANE K. HAWLEY' 3* Institute of ' and Departments ofBiology2 and Chemistry, University of Oregon, Eugene, Oregon 97403 Received 11 June 1990/Accepted 8 August 1990

Transcription from the adenovirus major late (ML) has previously been shown to pause or terminate prematurely in vivo and in vitro at a site within the first intron of the major late transcription unit. We are studying the mechanism of elongation arrest at this site in vitro to define the DNA sequences and proteins that determine the elongation behavior of RNA polymerase II. Our assay system consists of a nuclear extract prepared from cultured human cells. With standard reaction conditions, termination is not observed downstream of the ML promoter. However, in the presence of Sarkosyl, up to 80% of the transcripts terminate 186 nucleotides downstream of the start site. Using this assay, we showed that the DNA sequences required to promote maximal levels of termination downstream of the ML promoter reside within a 65-base-pair region and function in an orientation-dependent manner. To test whether elongation complexes from the ML promoter were functionally homogeneous, we determined the termination efficiency at each of two termination sites placed in tandem. We found that the behavior of the elongation complexes was different at these sites, with termination being greater at the downstream site over a wide range of Sarkosyl concentrations. This result ruled out a model in which the polymerases that read through the first site were stably modified to antiterminate. We also demonstrated that the ability of the elongation complexes to respond to the ML termination site was promoter specific, as the site did not function efficiently downstream of a heterologous promoter. Taken together, the results presented here are not consistent with the simplest class of models that have been proposed previously for the mechanism of Sarkosyl-induced termination.

Regulation of transcription is known to be an important tion system to study the mechanism of transcription termi- mechanism for altering gene expression during differentia- nation in vitro downstream of the adenovirus major late tion and development in eucaryotic cells. Much of this (ML) promoter. In vivo, transcription from the ML pro- regulation occurs at the level of initiation of transcription by moter pauses or terminates within intron 1 of the major late RNA polymerase II (pol II), the nuclear enzyme that tran- transcription unit at late but not early times during adenovi- scribes protein-encoding genes. Recently, research in a rus infection (29, 32). In vitro, extended pausing or termina- number of laboratories has demonstrated that pol II is also tion at this site is not observed under standard reaction subject to regulation after initiation by selective arrest of conditions when the ML promoter is transcribed in nuclear transcription elongation at specific sites within genes (re- extracts prepared from uninfected human cells. However, in viewed in reference 50). Perhaps the best evidence for such the of more than the regulation has been obtained through the study of expression presence Sarkosyl, 80% of transcripts of the c-myc gene in humans and mice. In those systems, initiated at the ML promoter indefinitely paused or termi- changes in the ability of transcription elongation to proceed nated at this site (16). past the 3' end of exon 1 have been shown to correlate with The finding that Sarkosyl promoted termination at a spe- changes in c-myc expression that occur during differentiation cific site downstream ofthe ML promoter was an outcome of and with the abnormal expression of c-myc in some Burkitt's experiments originally designed to test the usefulness of lymphoma cells (reviewed by C. A. Spencer and M. Grou- Sarkosyl as a means of blocking transcription initiation (16, dine, Adv. Cancer Res., in press). Evidence for regulated 17). Sarkosyl had previously been shown to prevent initia- blocks to transcription elongation has also been obtained for tion but to allow elongation by purified pol II in vitro at other cellular genes, including the mouse c-myb, hamster concentrations that dissociated most DNA-binding proteins c-fos, Drosophila hsp70, and human and mouse adenosine (14). Consistent with that observation, Sarkosyl was found deaminase genes and viral genes encoded by simian virus 40, to inhibit pol II transcription initiation in a nuclear extract polyomavirus, human adenovirus, minute virus of mice, and system as well but not to inhibit elongation generally (16). human immunodeficiency virus (2, 7, 11, 13, 18, 22, 27, 29, These initial observations have now been confirmed and 38, 41, 48). extended by a number ofgroups studying initiation in vitro at To date, most of the studies of potential attenuation sites the ML promoter, as well as at other class II promoters, and for pol II transcription have relied primarily on nuclear the usefulness of this approach for limiting transcription by run-on assays and isolation of prematurely terminated or pol II to a single round of initiations is well documented (4, processed transcripts from injected oocytes or virus-infected 5, 8, 15, 28, 47). Moreover, other groups have now demon- cells. Neither approach is amenable to detailed mechanistic strated Sarkosyl-dependent termination or pausing by pol II studies. We used a cell-free, promoter-dependent transcrip- at other sites corresponding to transcriptional attenuation sites in vivo (37, 38, 40). The method may, therefore, have a general usefulness in characterizing the elongation reaction * Corresponding author. as well. However, it remains to be determined whether the 5782 VOL. 10, 1990 TERMINATION IN VITRO BY RNA POLYMERASE II 5783 mechanism of induction of termination by Sarkosyl is the from pAdTr to the HindIII site ofplasmids pPT+ and pP+T, same at these various sites. respectively. To make plasmids pP+TT.2 and pP+T+T, we Because it is an unusually strong promoter in vitro, the first digested pAdT and pAdT+, respectively, with SmaI ML promoter has been studied extensively and was used in and PvuII to generate a DNA fragment containing all the the original isolation and characterization of transcription adenovirus sequences from each plasmid flanked by vector factors (10, 31, 42). For these reasons, and because of the DNA. The DNA fragment was isolated and inserted between high efficiency of termination we have observed, this homol- the promoter and sites in pP+T, using a BamHI ogous system is uniquely suited for the study of the proteins site that had first been filled in with the Klenow fragment of and DNA sequences responsible for termination by pol II in DNA polymerase I to make it compatible with the blunt- vitro. As a first step, we began to define the specific DNA ended restriction fragment. sequences that contribute to termination downstream of the To make the plasmid pMPT+, we first constructed the ML promoter and to test several simple models for the plasmid pM,0, which contains mouse 3-globin DNA from mechanism of Sarkosyl-induced termination. the ClaI site at -110 to the BamHI site at +475 ligated to the AccI and BamHI sites of pTZ18U. These 1-globin sequences MATERIALS AND METHODS were obtained from the plasmid pMH,8 (33; gift from R. Myers, University of California, San Francisco) and con- Plasmid constructions. The plasmid pMLH1, which con- tained an 8-bp BglII linker at +26. To construct pMPT+, tains adenovirus DNA sequences from -260 to +536 with pMIO was restricted at a PstI site immediately upstream of respect to the ML cap site, has been described previously the ,-globin sequences, and the 3' overhanging ends were (17). pMLH3, which was used as a source of promoter- or removed with T4 DNA polymerase. This DNA was then terminator-containing DNA during construction of several of restricted with BglII, and the resulting DNA fragment con- the plasmids described below, contains ML promoter se- taining ,B-globin sequences from -110 to +26 was ligated to quences from -260 to + 198 and was made by ligating a pAdT+ restricted with SmaI and BamHI. PstI-HindIII DNA fragment from pMLH1 to the PstI and Plasmids pPC28T and pPC246T were derivatives of plas- HindIII sites in the vector pTZ19U (U.S. Biochemicals). mids we obtained in making a series of 3' deletions of the The plasmid pPT was made in two steps. First, a DNA G-less cassette sequence in pMLC2AT (45). The construc- fragment containing adenovirus sequences from the HaeIII tion of those plasmids will be presented in greater detail site at + 153 to the HindIII site at + 193 was purified and elsewhere. The parent plasmids used in this study contained ligated to the vector pTZ18U (U.S. Biochemicals) restricted ML promoter DNA from -260 to + 10 followed by 28 or 246 with HincII and HindIII. The resulting plasmid was called bp of the original G-less cassette sequence inserted into the pAdT. Next, a 337-base-pair (bp) fragment was purified after vector pTZ18U. A HindIII fragment containing adenovirus digestion of pMLH3 with EcoRI and PvuII. The EcoRI site sequences from pAdTr was ligated to each parent plasmid at was located upstream of adenovirus sequences, and the a HindIII site located 4 bp downstream of the end of the PvuII site was at +33 with respect to the ML cap site. This G-less cassette. fragment was ligated to pAdT restricted with EcoRI and The dideoxy-chain termination DNA-sequencing method SmaI to create pPT. To make the plasmid pP+T, we of Sanger et al. (43) was used to confirm the DNA sequences digested pMLH3 with SstII and isolated a DNA fragment of all these plasmids except pP+TT.2, which contains two containing adenovirus sequences from -250 to + 131. The 3' universal primer sites. The structure of that plasmid was overhanging ends resulting from SstII digestion were re- verified by restriction analysis. Modified T7 DNA polymer- moved by using T4 DNA polymerase (30), and the fragment ase (Sequenase; U.S. Biochemicals) was used for the DNA was ligated to pAdT restricted with SmaI. Plasmid pPT+ sequencing, following the instructions of the supplier. was made in two steps. In the first step, pMLH3 was Nuclear extracts and . Nuclear extracts were digested with SstII and a fragment containing adenovirus prepared by the procedure of Dignam et al. (9), except that sequences from +132 to +198 and all the vector DNA was all buffers contained 20 mM Tris hydrochloride (pH 7.9 at isolated. The SstII ends were made flush with T4 DNA 4°C) instead of HEPES (N-2-hydroxyethylpiperazine-N'-2- polymerase, and the treated plasmid was restricted with ethanesulfonic acid). In the early phases of this work, HindIll. The resulting 65-bp DNA fragment containing ade- extracts were prepared from HeLa cells. We subsequently novirus sequences was ligated to pTZ18U restricted with used nuclear extracts prepared from Namalwa cells, a line of HinclI and HindIII to create the plasmid pAdT+. The human Burkitt's lymphoma cells (obtained from R. Roeder, second step of this construction, in which an ML promoter- Rockefeller University); all the experimental results shown containing DNA fragment was inserted upstream of the were obtained with these extracts. We observed no qualita- adenovirus terminator, was the same as the second step in tive differences in the two types of extracts for the studies the construction of pPT described above. described here, although we observed less variability in the Four plasmids were made which contained the ML pro- activity of the extracts prepared from Namalwa cells. moter and duplications of some ML downstream sequences. Namalwa cells were cultured at 37°C in Spinner flasks in Two of these constructions made use of a plasmid, pAdTr, Biorich medium (Flow Laboratories) supplemented with 2.4 which was obtained accidentally during construction of g of sodium bicarbonate per liter, 0.29 g of L-glutamine pAdT (described above). pAdTr resulted from ligation of (Sigma) per liter, Eagle minimal essential medium nonessen- two DNA fragments into Hindlll-restricted pTZ18U. One of tial amino acids (Flow), 100 IU of penicillin-streptomycin these fragments was adenovirus DNA from the HaeIII site at (Flow) per ml (100 Rg/ml), and 5% iron-supplemented bovine + 153 to the HindIII site at + 193. The other fragment was 13 calf serum (Hyclone). The cells were harvested at a density bp of DNA from the HindIlI site to an HaeIII site just of 2 x 106 to 3 x 106 cells per ml. outside the polylinker region of pUC13. These fragments Transcription reactions. Templates were prepared by di- were inserted into the vector in such a way that the adeno- gesting plasmid DNA with BglI (or, for pMLH1, SmaI). The virus DNA was flanked by HindlIl sites. Plasmids pPT+T protocol used for all transcription reactions included an and pP+TT.1 were made by ligation of the HindIII fragment initial incubation of template and nuclear extract for 60 min 5784 WIEST AND HAWLEY MOL. CELL. BIOL. at 30°C without nucleotides, followed by the addition of sum of the molar amounts of second terminated and runoff nucleotides and Sarkosyl and a second incubation at 30°C as transcripts. Percent termination was calculated by multiply- described below. The solution conditions for the first incu- ing these fractions by 100%. bation were as follows: 12 mM Tris hydrochloride (pH 7.9 at 4°C), 12% glycerol, 60 mM KCl, 0.1 mM EDTA (all contrib- RESULTS uted by the storage buffer for the extract), 20 mM HEPES (pH 8.4 at 25°C), 10 mM MgCl2, 30 ,ug of template DNA per Previous studies showed that adding Sarkosyl to a tran- ml, and 2.5 to 3 mg of protein per ml in a total volume of 20 scription reaction in vitro resulted in a marked reduction in ,ul. After this incubation, ATP, CTP, [a-32P]UTP, and phos- the amount of full-length transcript observed from the ML phocreatine (3 RI) were added, followed at 30-s intervals by promoter and the concomitant appearance of a shorter the addition of Sarkosyl (2 [lI) and GTP (2 ,ul). The final transcript estimated to be 186 nucleotides (nt) long (16). concentrations in the second incubation were as follows: When transcript production was monitored as a function of ATP and CTP, 600 ,uM; UTP, 25 ,uM, 4 to 5 ,uCi per reaction time after addition of nucleotides, the 186-nt transcripts were (800 Ci/mmol; Dupont, NEN Research Products); and phos- shown to accumulate at earlier times than full-length tran- phocreatine (Sigma), 10 mM. GTP was added to 25 or 600 scripts. In the presence of lower concentrations of Sarkosyl, ,M and Sarkosyl (International Biotechnologies, Inc.) was transcription paused at the same site, but the short tran- added to 0.05 or 0.3%, as described in the legend to each scripts did not persist. These results suggested that the figure. The concentrations of Sarkosyl used were determined 186-nt transcripts were produced by pausing or termination by titration of a 10% stock solution; 0.05% was found to of transcription rather than by processing of a longer tran- prevent reinitiation of transcription, while 0.3% was re- script. quired for maximum levels of termination. For the titrations We have not yet determined whether the 186-nt transcripts of ATP, CTP, and UTP, this part of the protocol was that accumulated in the presence of Sarkosyl paused indef- modified slightly, as detailed in the legend to Fig. 1. Reac- initely or were released from the template. However, we tions were stopped 44 min after GTP addition by the addition have not found any conditions that allowed these short of 50 RI of a stop mixture consisting of 10 mM EDTA, 0.1 M transcripts to be chased into longer products. Neither addi- sodium acetate (pH 5.5), 0.5% sodium dodecyl sulfate, and 1 tion of high concentrations of nucleotides nor dilution of mg of yeast RNA per ml. The quenched reactions were Sarkosyl, nor both, resulted in a reduction of the amount of extracted once with an equal volume of phenol-chloroform; the short transcripts once they were formed in the presence the organic phase was reextracted with 80 ,ul of stop mixture. of high concentrations of Sarkosyl (data not shown). For this The combined aqueous phases were precipitated in ethanol reason and to simplify the discussion that follows, we refer at -20°C. After centrifugation, the RNA pellets were dried to production of these transcripts as termination. under vacuum and suspended in 15 RI of 98% formamide Termination efficiency depends on GTP concentration. In plus dyes and electrophoresed on a 6% polyacrylamide gel the original experiments of Hawley and Roeder (16), the (30:0.8 acrylamide-bisacrylamide) containing 7 M urea. The labeled and, therefore, limiting nucleotide was GTP. We running buffer was 0.09 M Tris-0.09 M borate-2.5 mM subsequently found that when higher concentrations of GTP EDTA. The gel was dried under vacuum and exposed to were used, the amount of terminated transcript decreased. Kodak BB-5 film for 6 to 12 h at -80°C with an intensifying This observation prompted us to test the dependence of screen. termination on the concentration of each of the four nucle- Quantification of transcripts. Slices corresponding to tran- oside triphosphates. The template used for these analyses script bands were cut from the dried gel, using the autora- was the plasmid pMLH1, which contains adenovirus se- diogram as a guide. Slices of equal size above the transcript quences from -260 to +536 with respect to the start site of band were also cut from the gel and used to determine transcription from the ML promoter. Figure 1A shows the background radioactivity. The amount of radioactivity in general protocol used. Nuclear extract and pMLH1 DNA each gel slice was determined by scintillation counting in were incubated together at 30°C to permit formation of Ecolume scintillation fluid (ICN Biomedicals). preinitiation complexes. Three of the four nucleotides- The molar concentration of transcript (txpt) represented ATP, CTP, and UTP-were added to allow transcription to by each gel slice was calculated by the following equation: initiate but not elongate extensively. The nucleotide being - varied was added separately 30 s after the addition of the cpm in txpt slice cpm in background slice other two nucleotides. Sarkosyl (denoted by S in Fig. 1A) [txpt] = total cpm added to reaction mixture was then added to 0.3%, followed by the addition of the remaining nucleotide, GTP. Reactions were incubated to [UTP] in reaction mixture permit initiated complexes to elongate. RNA from each x was and Meth- no. of uridines per txpt reaction processed as described in Materials ods, and the transcripts were resolved by gel electrophore- where cpm is counts per minute. For templates with a single sis. The radioactivity incorporated into full-length 536-nt termination site, the fraction of terminated transcripts was transcripts and 186-nt terminated transcripts was measured calculated by dividing the molar amount of terminated for each reaction, and the percent termination was plotted as transcript by the sum of the molar amounts of terminated a function of the concentration of the nucleotide that was and runoff transcripts. For templates with two termination varied (Fig. 1B). sites, the fraction of terminated transcripts at the first site We found that the extent of termination was highly depen- was calculated by dividing the molar amount of transcript dent on the concentration of GTP in the reaction. More than corresponding to the first termination site by the sum of the 80% of the transcripts terminated at 25 ,uM GTP, whereas molar amounts of runoff transcript and transcripts termi- only about 15% terminated at 600 ,uM GTP. Replotting the nated at the first and second sites. The fraction of transcripts data as [GTP]/fractional readthrough versus [GTP] yielded a that terminated at the second site was obtained by dividing Km of 70 ,uM for GTP for readthrough of the termination site the molar amount of the second terminated transcript by the (data not shown). VOL. 10, 1990 TERMINATION IN VITRO BY RNA POLYMERASE II 5785

A NE, DNA 2 NTPs 1 NTP S GTP STOP I 1 O' 60' 61 1 105' 60.5' 6 1 .5' 100

80 c 0

60 O._E

L- 40 a)cJ a 201

4tM Nucleotide

FIG. 1. Dependence of termination efficiency on nucleotide concentration. The concentration of each of the four nucleoside triphosphates was varied individually in transcription reactions containing 0.3% Sarkosyl. (A) The protocol used for this analysis is shown. The concentration of each of the nucleotides when held constant was 600 ,uM, except for the a-32P-labeled nucleotide, which was 25 ,uM UTP in the ATP and CTP titration reactions and 25 ,uM CTP in the GTP and UTP titration reactions. Additional experimental details and the solution conditions are described in Materials and Methods. (B) Transcription reactions were processed and analyzed after gel electrophoresis as described in Materials and Methods. The bands corresponding to the full-length and terminated transcripts were cut from the gels, the amount of each transcript was determined, and the percent termination observed in each reaction was calculated (see Materials and Methods). This value was plotted as a function of the concentration of the nucleotide that was varied. Symbols: U, GTP; A, UTP; O, ATP; A, CTP.

The dependence of termination on GTP concentration terminated transcript from the wild-type template pMLH1 reflected a specific rather than a general dependence on (lanes 1 to 4) showed the expected dependence of termina- nucleotide concentrations. A small increase in termination tion on Sarkosyl and GTP concentrations. The production of efficiency was observed at the lowest UTP concentrations terminated transcripts was negligible when the Sarkosyl tested (Fig. 1B). However, varying CTP and ATP over the concentration was low, regardless of the GTP concentration same range of concentrations had no effect on the percentage (lanes 1 and 4). When a high concentration of Sarkosyl was of terminated transcripts. included in the reaction, the terminated transcript was DNA sequences required for efficient termination. To deter- observed at both GTP concentrations, although the amount mine the DNA sequences necessary for production of the of termination was much higher at 25 ,uM GTP (lane 3) than prematurely terminated transcript, we prepared a series of at 600 p.M GTP (lane 2). The percent termination in each deletion mutations that removed DNA base pairs between reaction was determined by measuring the radioactivity the promoter and the termination site (Fig. 2A). Each of incorporated into each transcript as described in Materials these templates was tested for the ability to promote termi- and Methods, and the results are shown in Table 1. nation at the site corresponding to + 186 in the intact The transcripts observed in reactions containing the de- adenovirus sequence (pMLH1). The sensitivity of the reac- leted templates are shown in lanes 5 to 16 of Fig. 2B. For tion to Sarkosyl and GTP concentrations was also tested in each of these templates, we observed a transcript of the size each case, because we assumed that if termination was expected if termination was occurring at the normal site occurring by the same mechanism with these different tem- (transcripts labeled t in lanes 7, 11, and 15). Moreover, these plates, the dependence on reaction conditions would be transcripts were observed in the highest amounts in the retained. The templates were transcribed under conditions reactions containing high Sarkosyl and low GTP, the same of low (0.05%) and high (0.3%) Sarkosyl and low (25 p.M) pattern as observed for the fully intact template, pMLH1. and high (600 ,uM) GTP. A Sarkosyl concentration of 0.3% However, only one of the deleted templates (PT+) showed a was determined by titration to result in maximum levels of termination efficiency comparable to that observed for 186-nt transcript, while 0.05% Sarkosyl limited transcription pMLH1. The maximum level of termination observed for the to a single round of initiations but did not result in significant templates PT and P+T was only about one-half that ob- accumulation of terminated transcript (data not shown; but served for the templates pMLH1 and PT+ (Table 1). see reference 16). For reasons we do not entirely understand, the total The transcripts observed for reactions with the intact and amount of promoter-specific transcription (the sum of the deleted templates are shown in Fig. 2B. Production of the runoff and terminated transcripts) observed in reactions 5786 WIEST AND HAWLEY MOL. CELL. BIOL.

TERMINATION SITE (+186) TEMPLATE A

.'U .53 I -260 33J -536 1 1 I I.

pMLHI m

PT T1 I 4 nt txpt I P 1~~~~~~~~~~~~~~~~~~ PT+ 1,, T+ |I | I I 11 04nt txpt I I I P+T LIEI 1 8 1 nt txpt

B 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 SARKOSYL L H H L L H H L L H H L L H H L GTP H H L L H H L L H H L L H H L L . _ A AM *a

a

^ -020* m;p ;z; - -~~4ot _~~~~~~~o * _ A w .f.:I- rC imm.

..M- f mm *__~~~~~~~ r -am_4

t-_. VOL. 10, 1990 TERMINATION IN VITRO BY RNA POLYMERASE II 5787

TABLE 1. Quantification of the experiment of Fig. 2 sequences changes the sequence of the RNA, and possibly % Termination also the dependence of the termination on nucleotide con- % Sarkosyl [GTP] (pLM) centrations, we have not ruled out the possibility that pMLH1 PT 1PT+ P+T termination at this site occurs under some conditions. How- 0.05 600 1 3 0 0 ever, we have tested a variety of reaction conditions, includ- 0.3 600 30 10 22 11 ing different limiting nucleotides, and have not yet detected 0.3 25 84 31 75 38 termination at the inverted site (D. K. Wiest, unpublished 0.05 25 3 6 9 2 data). Templates with tandem termination sites give rise to two terminated transcripts. To gain insight into the mechanism of containing a high concentration of Sarkosyl frequently ex- Sarkosyl-dependent termination downstream of the ML pro- ceeded that observed at lower Sarkosyl concentrations. One moter, we prepared templates that contained two termina- possible reason for this difference is that the elongation of tion sites in tandem. We reasoned that if Sarkosyl was the polymerases to the end of the template was more efficient directly affecting the ability of pol II to elongate past the at higher Sarkosyl concentrations. Consistent with that termination site, then we might expect that the probability of interpretation is the observation that shorter-than-full-length termination at the first and second sites would be the same transcripts were sometimes more prominent at lower Sarko- under all reaction conditions. However, if Sarkosyl was syl concentrations (for example, in lanes 5, 8, and 12 of Fig. disrupting an association between the polymerase and an- 2B). If these shorter transcripts represented elongation com- other protein, or between some protein and the DNA or plexes from the ML promoter, then we underestimated the RNA, then an intermediate Sarkosyl concentration might total transcription in those reactions and, consequently, generate a heterogeneous population of elongation com- overestimated the efficiency of termination under low Sar- plexes that exhibit different behaviors at the two sites. For kosyl conditions. The conclusion that termination efficiency example, elongation complexes that did not terminate at the on all the templates in this study is insignificant at 0.05% first site might also be more likely to read through the second Sarkosyl would be made even stronger by such an interpre- site. tation. We constructed three different templates containing two The experiment of Fig. 2 and Table 1 led to three conclu- termination sites located at different distances from the sions about the regions of DNA sequence contributing to the promoter and from each other, as diagrammed in Fig. 3A. function of the ML termination site. First, we concluded that The DNA fragments the termination sites corre- adenovirus DNA between +33 and +133 was not required containing for maximum termination under the experimental conditions sponded either to the fully functional site defined in the tested, because we observed the same level of termination experiment of Fig. 2 (sequences from + 133 to + 198), which for pMLH1 and PT+. Second, the reduced termination we refer to as the T+ site, or to the reduced efficiency site efficiency of two templates that lacked DNA sequences (sequences from +153 to +198), which we refer to as the T between +131 and +153 (PT and P+T) showed that base site. The sizes of the runoff and terminated transcripts pairs within that region were not required for termination but expected for each template are listed in Fig. 3A. We mea- did contribute to the efficiency. Third, sequences upstream sured the efficiency of termination in vitro at each of the two of + 153 did not appear to be required for correct positioning sites at low and high concentrations of Sarkosyl and low and of the 3' end of the terminated transcripts, as all templates high concentrations of GTP. We found that each of the showed termination at or within a few base pairs of the templates produced transcripts corresponding to both termi- expected sites. In addition, this experiment showed that the nation sites (labeled t1 and t2 in Fig. 3B). The amount of each termination efficiency was not significantly altered when the transcript was quantified and is presented as percent termi- distance of the termination site from the promoter was nation in Table 2. decreased. For each template, termination at both the first and second We also found that the DNA sequences required for sites showed the normal dependence on Sarkosyl and GTP termination functioned in an orientation-dependent manner. concentrations. However, for each of these templates, ter- We constructed a plasmid containing the DNA sequences mination efficiency at the second site was higher than that from + 153 to + 198 in an inverted orientation with respect to observed at the first site (Fig. 3B and Table 2). Termination the promoter. When this plasmid was used as a template, we at the second site was still completely dependent on the saw no terminated transcripts and no reduction in the addition of high concentrations of Sarkosyl to the reaction. amount of runoff transcript, even under conditions that These results were particularly surprising in that in all cases promoted >40% termination from the site in its normal the second site was the T sequence, which was less efficient orientation (data not shown). Since inversion of the DNA at promoting termination than the more extensive T+ site

FIG. 2. Transcription in vitro from major late templates deleted for downstream DNA sequences. (A) DNA templates used to localize sequences required for production of terminated transcripts (txpt) are shown. Sequences corresponding to adenovirus DNA are represented as open boxes. Each of the deleted templates contains a promoter element (P or P+) and a downstream element (T or T+) separated by 17 bp of polylinker sequences. The vertical, dashed, bold line indicates the position of the termination site. Also indicated for each template is the size expected for transcripts terminating at the site corresponding to + 186 in pMLH1. (B) Each of the templates was transcribed in vitro according to the protocol described in Fig. 1A, except that ATP, CTP, and UTP were added together, followed after 30 s by the addition of Sarkosyl to a final concentration of either 0.05% (L) or 0.3% (H). After an additional 30 s, GTP was added to concentrations of either 25 ,IM (L) or 600 ,IM (H). Transcripts produced in each reaction were resolved on a polyacrylamide gel as described in Materials and Methods. An autoradiogram of the gel is shown. Arrows indicate the runoff(r) and terminated (t) transcripts in each set of reactions. The reaction conditions are shown above the lanes. The templates used in each set of reactions were as follows: lanes 1 to 4, pMLH1; lanes 5 to 8, PT; lanes 9 to 12, PT+; lanes 13 to 16, P+T. 5788 WIEST AND HAWLEY MOL. CELL. BIOL.

SIZE OF TERMINATED A TRANSCRI DTS PT-T STeTI ST 2ND I tH * 4W _:

PTT.

I~ ` T

PTT.2

t-H 20OWt -7 , - , -

B I 2 3 4 5 6 7 8 9 10 1I 12 SARKOSYL L H H L L H H L L H H L OTP H H L L H H L L H H L L a0 ft Am ~

_ _- _= _ a _g_ am -44.1 _ _ .. w. s_;.

r -.&- e3wI soft

_-gD40-a __-

a 0 r--o e4abt t2-s r_ _~~~~~~AA

t2-4 a S

t1-o t1-

t2-0 0.4s

t1 -. aM

when present as the first or only site (e.g., in the experiment Sarkosyl concentrations. We also measured termination at of Fig. 2 and in this experiment). sites 1 and 2 in the PT+T template over a wide range of In the experiment shown in Fig. 3, termination efficiency Sarkosyl concentrations. At all concentrations tested, termi- at tandem termination sites 1 and 2 was tested at only two nation observed at the second (T) site was equal to or greater VOL. 10, 1990 TERMINATION IN VITRO BY RNA POLYMERASE II 5789

TABLE 2. Quantification of the experiment of Fig. 3 a bar graph (Fig. 5). The bars are patterned differently to % Termination indicate whether the sites represented are the first, second, or only sites in the template. This analysis showed that the % Sarkosyl [GTP] PI+T P+TT1 P+TT.2 DNA sequences within the T restriction fragment directed tl t2 tl t2 tl t2 termination with an efficiency of about 40% when present as the first or only site in the template. The only exception to 0.05 600 2 0 0 1 1 0 this pattern occurred with the template PC246T, in which the 0.3 600 16 32 8 39 8 19 0.3 25 62 77 22 87 39 65 site was located 315 bp from the promoter and which 0.05 25 5 0 2 5 3 2 promoted termination with about 80% efficiency. All the T sites that were located downstream of another termination site, either T or T+, showed a termination than that at the first (T+) site (data not shown). Taken efficiency in the range of 70 to 85%. In particular, with the together, these findings are not consistent with a simple PT+T template, in which the T site followed a T+ site, we model in which the probability of termination at each site observed a termination efficiency significantly higher than reflected the intrinsic properties of a homogeneous popula- was observed for sites located at a comparable or even tion of elongation complexes. greater distance from the promoter (compare the amount of Termination efficiency is increased when the T site is located termination observed for the 165-nt transcript with that farther from the promoter. One possible explanation for the observed for the 181- and 200-nt transcripts). This compar- finding that termination at the T site was more efficient when ison showed that the distance of the site from the promoter that site was placed downstream of another termination site could not be the sole contributing factor in the increased was that the distance ofthe site from the promoter influences termination efficiency at all the T sites present as second termination efficiency. To test this hypothesis, we prepared termination sites. Instead, we must consider the possibility a new template in which a single T site was placed down- that the presence of an upstream site was responsible for the stream of the ML promoter so that the predicted 3' end of high efficiency of termination at the downstream T sites in the RNA would occur at position +315, a greater distance the experiment of Fig. 3. Indeed, a direct effect of distance from the promoter than in any of the templates used in the from the promoter on termination efficiency is less certain, experiment of Fig. 3. The DNA separating the promoter and because we cannot rule out the possibility that the increased terminator in this template was a synthetic G-less cassette termination at the single site in the PC246T was similarly that lacked G residues in the transcribed strand. Only 10 bp dependent on a particular sequence within the G-less cas- of adenovirus sequence preceded the synthetic G-less cas- sette sequence upstream of that site. sette sequence; however, our experiments had not ruled out Termination efficiency is promoter specific. Results with the a requirement for transcribed DNA sequences upstream of deleted templates (Fig. 2) had indicated that sequences +33. Therefore, as a control for possible functional differ- between +33 and +133 were not required for and did not ences due to the removal of adenovirus DNA between +10 contribute to the efficiency of the Sarkosyl-induced termina- and +33, we also constructed an additional template which tion. These deleted templates did not test the requirement placed a T site downstream of the G-less cassette so that the for DNA sequences upstream of +33, including the pro- 3' end should occur at +97. These two templates are moter. To analyze the requirement for these sequences, we diagrammed in Fig. 4A. We transcribed these templates in tested the ability of transcription complexes initiated at the vitro and found that the 97-nt transcript was synthesized mouse P-globin promoter to terminate at the T+ site. A with the expected (45%) frequency in the presence of 0.3% schematic drawing of the template used in this experiment Sarkosyl and 25 ,uM GTP but that the 315-nt transcript was (MPT+) is shown in Fig. 6A. A 96-nt transcript was ex- produced at a significantly increased frequency (80%) (Fig. pected if termination occurred at the same site as observed 4B and Table 3). This result is consistent with the hypothesis downstream of the ML promoter. Figure 6B shows the than an increased distance between the promoter and termi- autoradiogram from one experiment in which the MAT+ nator could have contributed to the increased termination template was transcribed by using our four standard reaction efficiency observed at the downstream site for the templates conditions. We did not observe any transcripts correspond- containing tandem terminators. ing to the expected termination product under any of these To examine whether the increased termination efficiency reaction conditions. at the second of two tandem termination sites could be In other experiments, and upon longer exposures of the entirely due to an influence of distance of the second site autoradiograms, we have sometimes observed a heteroge- from the promoter, we compared the termination efficiencies neous cluster of transcripts of about the size expected for a measured at 0.3% Sarkosyl and 25 ,uM GTP for all templates terminated transcript from this template (data not shown). containing the T site. These data are compiled in the form of Production of these transcripts depends on the inclusion of

FIG. 3. Transcription in vitro from major late templates containing two termination sites. (A) The DNA templates used in this analysis contain a promoter element, P or P+, and two downstream elements, either T or T+, as indicated. The adenovirus DNA sequences contained within each element (represented by boxes in the figure) are as shown in Fig. 2A. The solid line represents plasmid sequences. Each of the downstream sequence elements has the same orientation with respect to the promoter as in the major late transcription unit. The sizes expected for transcripts which terminate within the T+ or T element at the site corresponding to + 186 in the major late transcription unit are listed. A more detailed description of each of these templates is presented in Materials and Methods. (B) Each of the templates described in panel A was transcribed by using the protocol and reaction conditions described in the legend to Fig. 2. The Sarkosyl concentrations used in each reaction are shown above the lanes as L (0.05%) or H (0.3%). The GTP concentrations used in each reaction are shown above the lanes as L (25 ,uM) or H (600 ,uM). Transcripts terminating at the first termination site are indicated by arrows labeled tl, and transcripts terminating at the second termination site are indicated with arrows labeled t2. The templates used in the reactions were as follows: lanes 1 to 4, PT+T; lanes 5 to 8, P+TT.1; lanes 9 to 12, P+TT.2. 5790 WIEST AND HAWLEY MOL. CELL. BIOL.

PC2aT A -260 '10 M P L LE755428T 97 nt terminated txpt PC246T -260 *IQ MLP |3-LE55(246) T 315 nt terminated txpt

B 1 2 3 4 5 6 7 8 SARKOSYL L H H L L H H L GTP H H L L H H L L

as- S

t- _e ease_*

am VOL. 10, 1990 TERMINATION IN VITRO BY RNA POLYMERASE II 5791

TABLE 3. Quantification of the experiment of Fig. 4 SIG.E % Termination FIRST % Sarkosyl [GTP] (pLM) SOD PC28T PC246T 100 0.05 600 0 0 0.3 600 15 31 0.3 25 45 79 80 0.05 25 2 0 c 0co c 60 0.3% Sarkosyl in the transcription reaction and is enhanced E by lowering the GTP concentration. Accurate quantification of such small amounts ofthe putative termination products is = 40 difficult, but an upper limit of about 20% was estimated for 0 the efficiency of termination downstream of the 3-globin 0 promoter under conditions in which >80% termination was a. 20, observed downstream of the ML promoter. 0 DISCUSSION 84 97 165 181 181 200 242 315 340 356 The sequence of adenovirus 2 from +133 to +198 with respect to the cap site of the ML promoter is shown in Fig. Transcript Size (nucleotides) 7. We these we the showed sequences, which referred to as FIG. 5. Comparison of the efficiency of termination at the T T+ site, to be both necessary and sufficient to promote element and the distance of the site from the promoter. The termination downstream of the ML promoter at the same efficiency of termination was compared for all the templates de- frequency as the intact template under a variety of experi- scribed in this study in which the T element was located downstream mental conditions in vitro. In particular, at 25 ,uM GTP and of the ML promoter. The percent termination indicated by each bar in the presence of 0.3% Sarkosyl, transcripts ending at this was the average of at least three separate measurements, except that site constituted 80% of the total transcripts observed from the efficiencies reported for the 97- and 356-nt transcripts were each the ML promoter. Removal of 20 bp of DNA from the 5' end measured only one time. Within each set of averaged numbers, of this sequence, generating what we called the T site, individual measurements varied by 8% or less from the calculated reduced the termination efficiency to about 40% under average. All reaction mixtures contained 0.3% Sarkosyl and 25 ,uM GTP. The bars are arranged in order of increasing length of the maximum termination conditions. terminated transcript for the site represented and are coded to Kessler et al. (24) have constructed 3' deletions of the ML indicate whether the site occurs as the only termination site in the site and have shown that termination (or, at least, pausing) template (SINGLE) or as the first or second site in a template still occurred when the deletion removed two of the five containing tandem termination sites. The templates represented, in consecutive T residues from + 182 through + 186. When only order of transcript size, were as follows: single sites, PT, PC28T, one T residue was retained, the site was still a weak pause P+T, and PC246T; first sites, P+TT.1 and P+TT.2; and second site. Their results, in combination with those we described sites, PT+T, P+TT.1, P+TT.2, and P+T+T. here, argue strongly that DNA sequences within and up- stream, but not downstream, of the run of Ts are the primary One possible function for DNA sequences upstream of the determinants of termination efficiency at the ML site. Thus, actual site of termination is that these sequences, when this site appears to differ from a minimal DNA sequence that transcribed, promote formation of an RNA structure. In- has been shown to promote termination by purified pol II in deed, Aloni and colleagues have proposed that RNA sec- vitro (23). This sequence, which occurs within the human ondary structure is important to the mechanism ofpausing or H3.3 gene, contains two clusters of Ts; termination termination at the ML site and other viral sites they have occurred within the first run of seven Ts. Nontranscribed studied (reviewed in reference 1). However, our finding that sequences, including the second run of six Ts and up to eight base pairs upstream of +133 were not required for termina- additional base pairs downstream of the termination site, tion in vitro rules out the involvement of several specific were shown to contribute to the termination efficiency. This potential secondary structures that Aloni and colleagues (24, difference in the location of the sequences required for 39) have proposed to be important to the termination mech- termination may reflect differences in the assay for termina- anism. If an RNA structure is required for pol II termination tion (Sarkosyl-induced termination in a nuclear extract at the at the ML site, then either the RNA bases involved must lie ML site, in contrast to termination by purified polymerase at entirely within the 50 bases upstream of the 3' end of the the histone site). Alternatively, these two sites may be RNA or alternative structures involving sequences within representative of different classes of sites that can block pol the vector DNA must be contributing to termination. In II elongation. regard to this latter possibility, we showed that the T+

FIG. 4. Transcription in vitro from templates with the T element at different distances from the promoter. (A) The DNA templates represented in the diagram contain either 28 or 246 bp of G-less cassette sequence between the ML promoter (-260 to + 10) and T element. The position corresponding to +186 in the ML transcription unit is at +97 or +315 for the templates PC28T and PC246T, respectively. txpt, Transcript; MLP, major late promoter. (B) The templates were transcribed in vitro under the four standard reaction conditions described in the legend to Fig. 2. The Sarkosyl concentrations used are indicated as L (0.05%) or H (0.3%). The GTP concentrations used are indicated as L (25 ,uM) or H (600 ,uM). The templates used in each reaction were as follows: lanes 1 to 4, PC28T; and lanes 5 to 8, PC246T. For each set of reactions, r indicates the runoff transcript (expected sizes of 256 and 474 nt) and t indicates the terminated transcript. 5792 WIEST AND HAWLEY MOL. CELL. BIOL.

tff!3-.+ L A-.- A,1 14 Wae :5

B 1 2 3 4 SARK;OSYL L H H L GTP H H L L

4-

S* A.

FIG. 6. Transcription in vitro from a template containing the adenovirus termination site and a heterologous promoter. (A) The template M,T+ contained mouse p-globin promoter sequences from -110 to +26 with respect to the cap site (represented as the box labeled MAP) and the adenovirus T+ element (shown in Fig. 2A). The two regions of DNA sequence were separated by 14 bp of vector polylinker DNA. A more detailed description of this plasmid is given in Materials and Methods. The plasmid DNA was restricted with BglI within vector sequences to allow production of a 258-nt runoff transcript. The expected size for a terminated transcript (txpt) was 96 nt. (B) The template MPT+ was transcribed in vitro as described in the legend to Fig. 2 under conditions of 0.05% (L) or 0.3% (H) Sarkosyl and 25 ,uM (L) or 600 ,uM (H) GTP. The position of the runoff transcript is indicated (r). The arrow labeled t indicates the expected position of a 96-nt terminated transcript. The amount of runoff transcript in lanes 1 to 4 ranged from 15 to 30 pM, with higher amounts at 0.3% Sarkosyl than at 0.05% Sarkosyl. Quantification of radioactivity in gel slices corresponding to 96 nt showed that the percentage of terminated transcripts under all conditions was less than 20%. VOL. 10, 1990 TERMINATION IN VITRO BY RNA POLYMERASE II 5793

T

5'GGTGATGCCTTTGAGGGTGGCCGCGTCCATCTGGTCAGAAAAGACAATCTTTTTGTTGTCAAGCTT I I 1 *133 *153 +186198 TERMINATION SITE FIG. 7. DNA sequences required for termination in vitro downstream of the ML promoter. The sequence of the nontemplate DNA strand from + 133 to +198 relative to the cap site of the adenovirus ML promoter is shown. The 5' boundaries of the two restriction fragments containing sequences in this region, termed T and T+ in this study, occurred at +153 and +133, respectively, as indicated. The arrow indicates the nucleotide at +186, the proposed site of termination in our experiments. fragment promoted termination with the same efficiency Sarkosyl concentrations. We are currently testing whether (within experimental error) when located downstream of varying the GTP concentration alters the length of the pause different DNA sequences. Inspection of the sequences under nontermination conditions and whether the sensitivity within the T+ fragment revealed limited potential for RNA to nucleotide concentration depends on the identity of the secondary structure, although several possible hairpins of base at +187. predicted stabilities in the range of -10 to -12 kcal/mol Termination at either the T or T+ site was absolutely could be found (unpublished observations). However, the T dependent on the addition of Sarkosyl to the transcription fragment showed even less potential for forming secondary reactions. Possible explanations for the mechanism of Sar- structure; and although the efficiency of termination within kosyl-induced termination include a direct alteration of the the T fragment was variable, the variability could not be elongation properties of pol II or the disruption of an correlated with the sequence of the flanking DNA. In two association (protein-protein or protein-nucleic acid) required templates, the contiguous DNA was a G-less cassette that for readthrough of the site. To begin to examine possible contained no G residues on the nontemplate strand, further mechanisms in greater detail, we placed two termination reducing the likelihood of an extended stable structure sites in tandem to measure the probability of termination by within RNA transcribed from vector sequences. Although an elongation complex that had already failed to terminate at these findings have not ruled out the possibility that an RNA a preceding site. This experiment was motivated by similar structure is required for termination at the ML site, they experiments by Roberts and his colleagues, who used this have put limits on the location and identity of the sequences approach to investigate the termination properties of bacte- contributing to such a structure. A more complete analysis rial RNA polymerase modified by the bacteriophage lambda of the sequences important to the termination efficiency may Q gene protein (52). They found that elongation complexes help to resolve whether RNA structure plays any role in the that read through one termination site also tended to escape mechanism of termination at this particular site. termination at a second site, indicating that those polymer- The ability of elongation complexes to read through either ases were stably modified to antiterminate. In contrast, we the T or T+ site was found to depend on the concentration found that pol II terminated at higher efficiency at the second of GTP in the transcription reaction but to be relatively site than at the first over a wide range of Sarkosyl concen- insensitive to the concentrations of the other nucleotides. trations. This result eliminated the possibility that the pop- We considered two classes of models to explain the speci- ulation of polymerases that transcribed through the first site ficity of this nucleotide dependence. First, GTP might be were immune to termination. The finding also reduced the bound and/or hydrolyzed by some protein in the extract that likelihood that Sarkosyl acted by directly affecting polymer- allows antitermination at this site. As a simple test of this ase or by disrupting an association that was rapidly in idea, we substituted GTP-y-S for GTP in the transcription equilibrium. Either of those mechanisms might have been reaction and measured the dependence of termination on the expected to produce a population of functionally homoge- concentration of this substrate analog. We found a titration neous elongation complexes, at least under some experimen- pattern identical to that observed with GTP, suggesting that tal conditions, leading to the same termination efficiency at hydrolysis of the P--y phosphodiester bond is not required both of the two tandem sites. Instead, the different termina- (D. K. Wiest, unpublished observations). Second, because tion efficiencies observed at the tandem sites indicated that G is the nucleotide incorporated at +187, the GTP concen- the elongation complexes were functionally heterogeneous. tration may affect the length of time the elongation complex Such heterogeneity could have resulted if more than one pauses at + 186, the proposed primary -site of termination protein were responsible for the elongation properties of the (Fig. 7), with a longer pause leading to a higher frequency of polymerase, or if the action of Sarkosyl was to some degree termination. There are precedents for this second model. site specific (e.g., the polymerase might have to be in a Several procaryotic attenuation sites have been shown to be certain configuration or paused at a specific site). preceded by strong transcriptional pause sites believed to be Several groups have proposed that the elongation protein necessary to couple the transcription termination efficiency SII (also called TFIIS) is a good candidate for a putative to the ofthe leader peptide (reviewed in reference antitermination factor (35-37). In reconstituted transcription 26). Pausing by the bacterial polymerase at several of these systems in vitro, the addition of SII has been shown to sites in vitro has been shown to be specifically influenced by decrease significantly the accumulation of a transcript from the concentration of the nucleotide that would be the next the ML promoter that probably corresponds to the termi- one added to the 3' end of the paused RNA (6, 12, 25). The nated transcript we observed in the presence of Sarkosyl (35, finding that pol II pauses extensively at the ML site at low 36). SII was also shown to promote readthrough of the Sarkosyl concentrations (16) raises the possibility that paus- histone H3.3 termination site when added back to an SII- ing is important to the mechanism of termination at higher depleted transcription system reconstituted with highly pu- 5794 WIEST AND HAWLEY MOL. CELL. BIOL. rified transcription factors (37). This action of SII was Vicki Chandler for a critical reading of the manuscript and members abolished by the addition of Sarkosyl. On the basis of these of the laboratory for useful advice and discussions. and other experiments, Kane and her co-workers have This work was supported by grant DMB-8703950 from the Na- argued that Sarkosyl does not affect the ability of pol II to tional Science Foundation and by a grant from the Medical Research Foundation of Oregon. D.K.W. was supported, in part, by a elongate normally, except by this inhibition of SII activity graduate training grant from the National Institutes of Health. (37). D.K.H. is a Searle Scholar of the Chicago Community Trust and a Although those experiments clearly demonstrated that SII Presidential Young Investigator of the National Science Founda- enhanced the ability of pol II to elongate through several tion. termination sites in vitro, the participation of other proteins in the elongation behavior of the polymerase in the nuclear LITERATURE CITED extract cannot be ruled out. Indeed, it is not surprising that 1. Aloni, Y., and N. Hay. 1985. Attenuation may regulate gene SII promoted readthrough of these sites, because SII has expression in animal viruses and cells. Crit. Rev. Biochem. been shown to increase the overall processivity of the 18:327-383. polymerase (21, 36, 44, 46, 49). The ML site has previously 2. Bender, T. P., C. B. Thompson, and W. M. Kuehl. 1987. been shown to be a pause site in vitro under reaction Differential expression of c-myb mRNA in murine B lymphomas in the site by a block to transcription elongation. Science 237:1473-1476. conditions that result quantitative readthrough of 3. Bentley, D. L., and M. Groudine. 1988. Sequence requirements (16). It is likely that pausing is an important part of the for premature termination of transcription in the human c-myc termination mechanism and that SII decreases this pausing. gene. Cell 53:245-256. The finding that termination at the ML site was much less 4. Burton, Z. F., M. Killeen, M. Sopta, L. G. Ortolan, and J. efficient downstream of the ,3-globin promoter than down- Greenblatt. 1988. Rap3O/74: a general initiation factor that binds stream of the ML promoter argues against a simple model in to RNA polymerase II. Mol. Cell. Biol. 8:1602-1613. which SII is the only important determinant ofthe elongation 5. Cai, H., and D. S. Luse. 1987. Transcription initiation by RNA characteristics of the polymerase. Although one could argue polymerase II in vitro: properties of preinitiation, initiation, and that the extent to which pol II associated with SII was elongation complexes. J. Biol. Chem. 262:298-304. influenced by promoter or early transcribed sequences, we 6. Chan, C. L., and R. Landick. 1989. The Salmonella typhimu- rium his leader region contains an RNA hairpin-depen- could not then explain why termination downstream of both dent transcription pause site: mechanistic implications of the promoters was completely dependent on Sarkosyl, if the effect on pausing of altered RNA hairpins. J. Biol. Chem. dissociation of SII was the only direct effect of Sarkosyl. 264:20796-20804. We do not yet know whether the failure to terminate 7. Chinsky, J. M., M.-C. Maa, V. Ramamurthy, and R. E. Keliems. downstream of the P-globin promoter reflected the absence 1989. Adenosine deaminase gene expression: tissue-dependent of some component required to terminate or the presence of regulation of transcriptional elongation. J. Biol. Chem. 264: some component that made termination more difficult. If the 14561-14565. pol II that initiated at the ML promoter acquired a propen- 8. Conaway, R. C., and J. W. Conaway. 1988. ATP activates sity to terminate, only sequences downstream to + 10 appear transcription initiation from promoters by RNA polymerase II in a reversible step prior to RNA synthesis. J. Biol. Chem. to have been necessary, because those were the only adeno- 263:2962-2968. virus sequences present in the PC28T and the PC246T 9. Dignam, J. D., R. M. Lebovitz, and R. G. Roeder. 1983. templates, both of which showed the expected (or greater Accurate transcription initiation by RNA polymerase II in a than expected in the case of PC246T) termination efficiency soluble extract from isolated mammalian nuclei. Nucleic Acids at the T site. Res. 11:1475-1489. Promoter-specific utilization of termination or RNA-proc- 10. Dignam, J. D., P. L. Martin, B. S. Shastry, and R. G. Roeder. essing signals has previously been demonstrated in vivo. In 1983. Eukaryotic gene transcription with purified components. the best-studied example, transcription initiated by pol II at Methods Enzymol. 101:582-598. the and U2 was shown to to a 11. Evans, R., J. Weber, E. Ziff, and J. E. Darnell. 1979. Premature Ul gene promoters respond termination during adenovirus transcription. Nature (London) termination or processing site called a 3' box located down- 278:367-370. stream of those genes (19, 53). In contrast, when the 12. Fisher, R. F., A. Das, R. Kolter, M. E. Winkler, and C. promoters for several protein-encoding genes were fused to Yanofsky. 1985. Analysis of the requirements for transcription the small nuclear RNA genes, transcription proceeded pausing in the operon. J. Mol. Biol. 182:397-409. through the 3' box (20, 34). More recently, Groudine and his 13. Fort, P., J. Rech, A. Vie, M. Piechaczyk, A. Bonnieu, P. colleagues (3, 51) have shown that the ability of elongating Jeanteur, and J.-M. Blanchard. 1987. Regulation of c-fos gene pol II to respond in vivo to two transcriptional blocks within expression in hamster fibroblasts: initiation and elongation of the human c-myc gene appears to depend, in part, on the transcription and mRNA degradation. Nucleic Acids Res. 15: from which is initiated. it is 5657-5667. promoter transcription Thus, 14. Gariglio, P., J. Buss, and M. H. Green. 1974. Sarkosyl activation likely not only that recognition of many eucaryotic termina- of RNA polymerase activity in mitotic mouse cells. FEBS Lett. tion and processing sites will be shown to be promoter 44:330-333. specific but also that this promoter specificity will prove to 15. Hai, T., M. Horikoshi, R. G. Roeder, and M. R. Green. 1988. be important to the mechanism of regulation in vivo. Our Analysis of the role of the transcription factor ATF in the finding that termination at the ML site in vitro was promoter assembly of a functional preinitiation complex. Cell 54:1043- specific will now allow systematic characterization of the 1051. proteins and DNA sequences that determine this specificity 16. Hawley, D. K., and R. G. Roeder. 1985. Separation and partial at this site. characterization of three functional steps in transcription initi- ation by human RNA polymerase II. J. Biol. Chem. 260:8163- 8172. ACKNOWLEDGMENTS 17. Hawley, D. K., and R. G. Roeder. 1987. Functional steps in transcription initiation and reinitiation from the major late We thank Ann Seifried for technical assistance and for construc- promoter in a HeLa nuclear extract. J. Biol. Chem. 262:3452- tion of many of the plasmids used in this study and Fenella Raymond 3461. for maintenance of the cultured cells. We thank Barbara Hoopes and 18. Hay, N., H. Skolnik-David, and Y. Aloni. 1982. Attenuation in VOL. 10, 1990 TERMINATION IN VITRO BY RNA POLYMERASE II 5795

the control of SV40 gene expression. Cell 29:183-193. specific transcription by mammalian RNA polymerase II: tran- 19. Hernandez, N. 1985. Formation of the 3' end of Ul snRNA is scription factor IIS stimulates elongation of RNA chains. J. directed by a conserved sequence located downstream of the Biol. Chem. 262:3331-3337. coding region. EMBO J. 4:1827-1837. 37. Reines, D., M. J. Chamberlin, and C. M. Kane. 1989. Transcrip- 20. Hernandez, N., and A. M. Weiner. 1986. Formation of the 3' end tion elongation factor SII (TFIIS) enables RNA polymerase II to of Ul snRNA requires compatible snRNA promoter elements. elongate through a block to transcription in a human gene in Cell 47:249-258. vitro. J. Biol. Chem. 264:10799-10809. 21. Horikoshi, M., K. Sekimizu, and S. Natori. 1984. Analysis of the 38. Resnekov, O., and Y. Aloni. 1989. RNA polymerase II is capable stimulatory factor of RNA polymerase II in the initiation and of pausing and prematurely terminating transcription at a pre- elongation complex. J. Biol. Chem. 259:608-611. cise location in vivo and in vitro. Proc. Natl. Acad. Sci. USA 22. Kao, S.-Y., A. F. Calman, P. A. Luciw, and B. M. Peterlin. 1987. 86:12-16. Anti-termination of transcription within the long terminal repeat 39. Resnekov, O., E. Ben-Asher, E. Bengal, M. Choder, N. Hay, M. of HIV-1 by tat gene product. Nature (London) 330:489-493. Kessler, N. Ragimov, M. Seiberg, H. Skolnik-David, and Y. 23. Kerppola, T. K., and C. M. Kane. 1990. Analysis of the signals Aloni. 1988. Transcription termination in animal viruses and for transcription termination by purified RNA polymerase II. cells. Gene 72:91-104. Biochemistry 29:269-278. 40. Resnekov, O., M. Kessler, and Y. Alowi. 1989. RNA secondary 24. Kessler, M., E. Ben-Asher, and Y. Aloni. 1989. 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