MOLECULAR AND CELLULAR BIOLOGY, Sept. 1993, p. 5159-51670270-7306/93/095159-09$02.00/0Copyright ©) 1993, American Society for Microbiology

Termination and Pausing of RNA Polymerase II Downstreamof Yeast Polyadenylation SitesLINDA E. HYMANt AND CLAIRE L. MOORE*

Department ofMolecular Biology and Microbiology, Tufts University Health Sciences Campus,136 Harrison Avenue, Boston, Massachusetts 02111-1800

Received 15 March 1993/Returned for modification 20 May 1993/Accepted 27 May 1993

Little is known about the transcriptional events which occur downstream of polyadenylation sites. Althoughthe polyadenylation site of a gene can be easily identified, it has been difficult to determine the site oftranscription termination in vivo because of the rapid processing of pre-mRNAs. Using an in vitro approach,we have shown that sequences from the 3' ends of two different Saccharomyces cerevisiae genes, ADH2 andGAL7, direct transcription termination and/or polymerase pausing in yeast nuclear extracts. In the case of theADH2 sequence, the RNA synthesized in vitro ends approximately 50 to 150 nucleotides downstream of thepoly(A) site. This RNA is not polyadenylated and may represent the primary transcript. A similarly sizednonpolyadenylated [poly(A)-] transcript can be detected in vivo from the same transcriptional template. AGAL7 template also directs the in vitro synthesis of an RNA which extends a short distance past the poly(A) site.However, a significant amount of the GAL7 RNA is polyadenylated at or close to the in vivo poly(A) site.Mutations of GAL7 orADH2 poly(A) signals prevent polyadenylation but do not affect the in vitro synthesis ofthe extended poly(A)- transcript. Since transcription of the mutant template continues through this region invivo, it is likely that a strong RNA polymerase II pause site lies within the 3'-end sequences. Our data supportthe hypothesis that the coupling of this pause site to a functional polyadenylation signal results in transcriptiontermination.

The synthesis of eukaryotic mRNAs requires accuratetranscription initiation, subsequent elongation of the tran-script beyond the polyadenylation site, and, finally, cleavageof the primary transcript and transcription termination. Allof these points are used by the cell to regulate mRNAsynthesis. The mechanisms of transcription initiation andmRNA polyadenylation are complex. Many of the factorsresponsible for these events, as well as their interaction witheach other and with the DNA template or primary transcript,have been precisely defined (for reviews, see references 13,28, 29, and 40). On the other hand, transcription termination,which is defined as the release of the transcription complexand the nascent RNA from the template, is arguably the leastwell understood aspect of eukaryotic mRNA transcription,yet it is extremely important in that readthrough transcrip-tion into adjacent genes can have undesirable consequences.For example, it is known that such transcription into adownstream promoter can cause promoter occlusion anddecrease transcription initiation from that promoter (18, 27).Potential problems could also arise when two promoters faceeach other if convergent transcription led to the formation ofRNA duplexes. In addition, premature termination is impor-tant as a regulatory mechanism for the expression of severalcellular and viral genes (38).

Several models have been proposed to explain how RNApolymerase II stops elongation and releases the nascenttranscript (for reviews, see references 20 and 28). One modelproposes that the primary event directing termination maybe some aspect of the polyadenylation process. This is basedon the observation that mutations in a poly(A) signal preventtermination. In one version of this model, exonucleolytic

* Corresponding author.t Present address: Department of Biochemistry, Tulane Univer-

sity Medical School, New Orleans, LA 70112.

degradation of the uncapped 3' cleavage product wouldcause the transcription complex to disengage from its tem-plate (10). Alternatively, the transcription complex could bemodified in some way after transcription of a poly(A) site(22) so as to destabilize the polymerase and cause it toterminate transcription. In this case, termination could be a

consequence of the polyadenylation machinery and pro-voked, for example, by assembly of a processing complexonto the nascent RNA chain. On the other hand, thepolyadenylation signal sequences may be bifunctional innature and work at the RNA or DNA level to causetermination in a manner independent of polyadenylation.While these models require that a functional poly(A) se-

quence be transcribed, additional sequences which cause thepolymerase to slow down or pause may also be involved.Thus, a strong signal for mRNA 3'-end formation mightconsist of a functional poly(A) site followed by a polymerasepause site (3, 28).

In contrast to poly(A) site-dependent termination, a se-

quence downstream of the poly(A) site in the gastrin genehas been identified which causes transcription terminationregardless of the presence of a poly(A) site (35). Thus, insome cases, RNA polymerase II may stop in the absence ofmRNA polyadenylation. Pausing or termination may also bedue to a steric block, such as a protein binding to DNA (9,11, 12).

Precursor transcripts are rapidly cleaved and polyadeny-lated in vivo, and the downstream cleavage product issubsequently degraded. This has made it difficult to studythe relationship between polyadenylation and transcriptiontermination. While the technique of run-on transcription inisolated nuclei has been used to define the site of transcrip-tion termination for some eukaryotic genes (for an example,see reference 12), it is not a system which is easily amenableto experimental manipulation.

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Because of the compact nature of the Saccharomycescerevisiae genome, it is likely that transcription terminationin this organism is an efficient process and takes place fairlyclose to the poly(A) site. Indeed, indirect evidence suggeststhat transcription termination occurs within approximately100 nucleotides (nt) of yeast poly(A) sites (25, 33). Toestablish a better system in which to study transcriptiontermination and determine its role in mRNA 3'-end forma-tion, we have investigated whether termination downstreamof poly(A) sites can be observed in yeast extracts competentfor correct RNA polymerase II transcription initiation. Wehave analyzed two yeast genes in vitro and detected RNAspecies with the characteristics expected for products oftranscription termination or pausing.

MATERIALS AND METHODS

Reagents. All restriction enzymes, modifying enzymes,and DNA size standards were purchased from New EnglandBioLabs and used according to the manufacturer's direc-tions. Radioactive nucleotides were from DuPont-NEN Re-search Products; RNase T2 was from Bethesda ResearchLaboratories. The nylon transfer membrane was from Mi-cron Separations Inc.

Plasmids. The construction of plasmid pLlOl has beendescribed before (15, 26, 39). To create pL501, plasmidpGAL7-1 (8) was digested with HincIl and SmaI, and the180-nt fragment containing the GAL7 poly(A) site was li-gated to pHZ18A2SMA cut with SmaI (15). Plasmid pL502was made in the same way as pL501 except that the startingplasmid, pGAL7-3, contained a 13-nt deletion, as describedby Chen and Moore (8). Radiolabeled RNAs were made byusing either T7 or T3 RNA polymerase and linearizedpGAL7-1 digested with HindIlI or Hinfl for antisense probesand AvaI for sense RNA or pT7T3ADH2 digested withEcoRI for antisense probes.

Yeast extracts and reaction conditions. Yeast nuclear ex-tracts were prepared by the methods of Lue and Kornberg(23) with the modifications of Berger et al. (5). Threedifferent preparations of nuclear extracts were used, and theoptimal protein concentration was determined for each ex-tract. Protein concentrations were determined by the Brad-ford assay (Bio-Rad) (extracts were at approximately 40mg/ml, with bovine serum albumin as a standard). Transcrip-tion reactions were carried out as described by Berger et al.(5) except that the GAL4-VP16 protein was omitted and thereaction time was increased to 20 min. Yeast whole-cellextracts were prepared by previously published procedures(7, 8), and the reaction was carried out as described by Chenand Moore (8).RNA analysis. Total RNA from the in vitro transcription

reaction was extracted twice with phenol-chloroform-isoamyl alcohol (24:24: 1) and precipitated with 2 volumes ofethanol. Total RNA from yeast cells was prepared as de-scribed by Ausubel et al. (4). RNase protection analysis wasdone by the method of Hart et al. (14). Northern (RNA) blotanalysis was performed after gel electrophoresis on 1.5%agarose-formaldehyde gels. Polyadenylated [poly(A)+]RNA was selected by hybridization to poly(U)-Sephadex(Bethesda Research Laboratories) as described by Mooreand Sharp (24) except that the 10% formamide wash wasomitted. RNase H treatment was performed as described byHyman and Wormington (16) except that 20 ,ug of total RNAisolated from yeast cells was used. DNA size standards wereprepared by filling in 5' overhangs from MspII-cut pBR322with [32P]CTP and Eschenchia coli Klenow enzyme. RNA

size standards were made by transcribing plasmidpT7T3ADH2 or pT7T3ADH2L (which contains an addi-tional 300 nt from the coding region of the ADH2 gene) withT3 after digestion with restriction enzyme BstBI, SspI,NheI, or HindlIl to produce antisense transcripts of 180,340, 400, and 600 nt, respectively.Yeast strains and methods. The S. cerevisiae strain used

for all in vivo RNA studies was DB745 (AMTa ade2 leu2ura3). The strain used for the nuclear extract was BJ296(A Ta/MATTa prcl-126/prcl-126 pep4-3/pep4-3 prbl-1122/prbl-1122 canl-llcanl-l), and the strain used for the whole-cell extracts was 1097/930 (MATa/MA Tbt leu2/+ trpl/trp/jprbl-1122/prbl-1122 pep4-3/pep4-3 prcl-407/prcl-407 his+). All yeast culture methods used were those described bySherman et al. (36).

RESULTS

Transcription termination occurs within yeast 3'-end se-quences in vitro. We previously demonstrated that sequencesfrom the 3' end of the alcohol dehydrogenase 2 (ADH2) genecan direct proper 3'-end formation when placed upstream ofa reporter gene (15). In vivo, transcripts derived from thefusion gene plasmid pLlOl (Fig. 1A) (15) are polyadenylatedat precisely the same site as the endogenousADH2 mRNA.From this result, we concluded that the sequences requiredfor transcription termination and/or cleavage and polyade-nylation must lie within the ADH2 insert. Since the role oftranscription termination in forming the 3' end of this fusiontranscript could not easily be addressed in vivo, we decidedto examine the transcription of the fusion gene plasmid invitro. The experiments described in this report were donewith yeast nuclear extracts, which have been shown byothers to accurately initiate transcription from RNA poly-merase II promoters (5, 23).

Plasmid pLlOl was incubated with nuclear extracts underconditions which produce transcripts initiated from theCYC1 promoter on the plasmid. We primarily used Northernblotting techniques for analysis of RNAs. We found othermethods for analysis of RNA to be problematic. For exam-ple, direct incorporation of radiolabeled nucleotides into denovo-synthesized RNAs was not possible because of thelarge amount of label incorporated into nonspecific high-molecular-weight RNAs. Nuclease protection techniqueswith either S1 or RNA T2 nuclease sometimes led to artifac-tual products because of the long A/U regions commonlyfound in yeast 3' untranslated regions. Northern blot analy-sis, on the other hand, had sufficient sensitivity and resolu-tion to characterize the RNA made in vitro and did not havethe limitations of the other techniques. An example of theNorthern blot analysis is shown in Fig. 1B.RNA synthesized in vitro revealed a small RNA of ap-

proximately 350 nt which contained ADH2 sequences (Fig.1B, lane 1). The RNA made in vitro (Fig. 1B, lane 1) wasslightly larger than theADH2 RNA made in vivo from pLlOl(Fig. 1B, lane 4). Because the cells were not grown inmedium containing ethanol, no RNA transcripts were de-tected from the endogenousADH2 gene (31). RNA synthesisin the in vitro reaction is dependent on the presence of theexogenous template, and it is not made in the absence ofnucleoside triphosphates (Fig. 1B, lanes 2 and 3). The size ofthe in vitro RNA suggested that sequences which specify3'-end formation, either termination or processing, werebeing recognized in the extracts.To determine whether a different 3'-end sequence could

yield truncated transcripts in vitro, the ADH2 sequence in

MOL. CELL. BIOL.

YEAST POLYMERASE II TRANSCRIPTION TERMINATION 5161

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1 2 3 4 5 6 7 8 9 1 2 3FIG. 1. Transcription of yeast fusion genes in vitro and in vivo. (A) Schematic representation of transcription templates. Plasmid pLlOl

carries a fusion gene that is under control of the GAL upstream activator sequence and driven by the CYCI promoter. The ribosomal protein(rp) 51 untranslated region and an AUG codon, followed by the 5' splice site and a shortened intron and 3' splice site, were fused in frameto the lacZ gene to create the parent plasmid pHZ18A2 (26, 37). The 3' end of the ADH2 gene (340 nt) was inserted at a unique restrictionsite in the intron (15). This sequence is on a high-copy-number plasmid and contains the URA3 gene for selection in yeast cells. Plasmid pL401is identical to pLlOl except that a T residue 73 nt upstream of the poly(A) site has been changed to a G residue. Plasmid pL501 contains 180nt from the 3' end of the GAL7 gene in place of theADH2 sequences in pLlOl. Plasmid pL502 is identical to pL501 except that it containsa 13-nt deletion in the GAL7 sequence (see text). p(A), poly(A) site. (B) Northern blot analysis ofRNA synthesized in vitro and in vivo. TotalRNA from transcription reactions with pLlOl (lanes 1 and 2) or pL501 (lanes 6 and 7) as a template or reactions to which no exogenous DNAwas added (lanes 3 and 8) was analyzed on 1.5% agarose gels containing formaldehyde. These products were compared with those from 10pg of total RNA from strains harboring plasmid pLlOl (lane 4) or pL5O1 (lane 9). Lane 5 contains RNA size markers of 180, 340, and 600 nt.The blot was hybridized to antisense probes forADH2 (lanes 1 to 5) or GAL7 (lanes 6 to 9). NTPs, nucleoside triphosphates. (C) Effect ofa-amanitin on transcription of pL501 in vitro. RNA was analyzed by Northern blot hybridization as described for panel B. Lane 1 shows theproducts of a transcription reaction with pL501 as the template; lane 2, transcription in the absence of a template; lane 3, an in vitro reactionidentical to the one in lane 1 except for the addition of a-amanitin (A) to 10 pg/ml.

pLlOl was replaced with a 182-nt fragment containing theGAL7 poly(A) site (Fig. IA, plasmid pL501). This fragmentcontains 69 nt of sequence upstream of the GAL7 poly(A)site and 113 nt of downstream sequence and directs GAL73'-end formation in vivo (Fig. 1B, lane 9). In vitro, the majortranscript produced from plasmid pL501 is approximatelythe same size as the RNA derived from this plasmid in vivo(Fig. 1B, compare lanes 6 and 9). As with the ADH2sequence, this RNA is also dependent on the exogenouslyadded plasmid template and on the presence of nucleosidetriphosphates (Fig. 1B, lanes 7 and 8). In addition, little RNAis produced in vitro when a-amanitin is added to the reactionmix at a concentration which inhibits RNA polymerase II(Fig. 1C). These results suggest that the RNA synthesizedfrom the CYCI promoter of the plasmid by RNA polymeraseII in vitro terminates within the ADH2 or GAL7 3'-endsequence. We have not determined the efficiency of tran-scription termination within the test sequences. However,after longer exposure of the autoradiograms shown in Fig.1B, it is clear that a significant amount of RNA is producedin vitro which does not terminate immediately downstreamof the poly(A) sites used in vivo. These readthrough tran-scripts are generally not resolved as specific bands onNorthern blots but appear as a heterogeneous smear.

In vitro transcription products contain sequences beyondyeast poly(A) sites. The Northern blot analysis, althoughhighly sensitive, did not allow us to determine the precise 3'

end of the RNA made in vitro. To investigate this, we usedan RNase protection strategy. A radiolabeled antisenseRNA for the ADH2 sequence was hybridized to total RNAfrom yeast cells containing pLlOl, to RNA made in vitrowith pLlOl as a template, and to an in vitro reaction mix inwhich the template was omitted. After treatment with RNaseT2, the protected fragments were analyzed on a denaturinggel (Fig. 2). The probe, which is 360 nt long, begins 170 ntupstream of the mapped poly(A) site and extends to aposition 190 nt downstream of the site. Because the probecorresponds to the genomic sequence, it will not protect thepoly(A) tail. The in vivo RNA yields two protected bands of150 to 160 nt (Fig. 2, lane 4). It is clear from this fine-structure analysis that the RNA made in vitro does notterminate at the in vivoADH2 poly(A) site (Fig. 2, comparelanes 3 and 4). Instead, a complex set of transcripts areproduced. The strongest band represents complete protec-tion of the probe (arrow) and corresponds to heterogeneousreadthrough RNA extending at least 190 nt beyond thepoly(A) site. It is clear that the majority of the transcriptsproduced in vitro do not terminate within the ADH2 se-quence, and therefore the efficiency of termination is low.The shorter RNase-protected products are 50 to 150 ntlonger than the same RNAs made in vivo (Fig. 2, lanes 3 and4) and most likely correspond to the prominent band de-tected by the Northern blot analysis. Two major species areobserved in this shorter class and have 3' ends approxi-

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5162 HYMAN AND MOORE

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1 2 3 4FIG. 2. Fine-structure mapping of pLlOl RNA made in vitro com-

pared with RNA made in yeast cells. Total RNA from a transcriptionreaction to which no exogenous template was added (lane 2), RNAfrom a transcription reaction which contained pLlOl as a template(lane 3), or 10 p,g of RNA from yeast cells harboring plasmid pLlOl(lane 4) was hybridized to an antisenseADH2 probe. The hybrids weretreated with RNase T2, and protected fragments were analyzed on a6% polyacrylamide-8.3 M urea gel. Lane 1 contains DNA markers(sizes shown in nucleotides). Arrow points to the fully protected probe,asterisks indicate major bands derived from RNAs with 3' ends withinthe ADH2 sequence, and the triangle marks protected fragmentscorresponding to the ends at the in vivo poly(A) site.

mately 50 and 150 nt downstream of the poly(A) site (Fig. 2,lane 3, asterisks).A comparable analysis was not possible for the GAL7 in

vitro-synthesized RNA. Because of the high A/U nucleotidecomposition of this sequence, it has been difficult to map theGAL7 3' ends by nuclease protection analysis (34). How-ever, as discussed below, Northern blot hybridization withprobes specific to the region downstream of the GAL7poly(A) site demonstrates that some of the GAL7 in vitrotranscripts also extend past the poly(A) site.

Transcription products containing the ADH2 3' end are notpolyadenylated. The differefice in size between the in vitro-transcribedADH2 RNA and the same RNA made in vivo isnot due to differences in transcription initiation sites, asprimer extension analysis showed that the same start sitesare used for both RNAs (data not shown). Therefore, themost likely explanation for the size change is a difference atthe 3' end of the transcripts. One possibility is that the invitro transcripts are not polyadenylated [poly(A)-] andrepresent the uncleaved RNA precursor. This would not beselected by poly(U)-Sephadex or oligo(dT) chromatography.Another possibility is that the transcripts are polyadenylatedbut at a different position than the in vivo RNA. Total RNAfrom an in vitro transcription reaction with plasmid pLlOl asthe template was subjected to poly(A) selection, and theRNA was analyzed on Northern blots. As shown in Fig. 3,even after long exposures of the autoradiogram, it is clearthat the in vitro pLlOl-derived transcripts are not polyade-nylated (Fig. 3, lane 2). In contrast, theADH2 RNA made invivo is retained by the poly(U)-Sephadex beads (Fig. 3, lane5). This suggests that the RNA generated in the in vitrotranscription system is a product of transcription terminationand could represent the uncleaved precursor.

It is formally possible that an unstable pre-mRNA is

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FIG. 3. pLlOl RNA made in vitro is not polyadenylated. TotalRNA from a standard transcription reaction was hybridized topoly(U)-Sephadex beads to select any RNA containing a poly(A) tailgreater than 10 nt in length. Fractionated RNA was analyzed afterelectrophoresis on 1.5% agarose-formaldehyde gels by Northernblot hybridization with an antisense ADH2 probe. Lane 1 containsunfractionated RNA made in vitro. Lane 2 represents RNA whichbound to the poly(U) beads, and lane 3 contains RNA which did notbind to poly(U)-Sephadex. Lanes 4, 5, and 6 contain 5 ,ug of RNAfrom yeast cells carrying plasmid pLlOl and represent unfraction-ated (T), poly(A)+, and poly(A)- RNA, respectively.

synthesized in vitro and that this precursor is processed to anintermediate form corresponding to the species that we haveanalyzed. We have not been able to address this possibility byclassical pulse-chase experiments. However, the time courseof the reaction (Fig. 4A) did not reveal pre-RNAs appearing

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1 2 3FIG. 4. RNA made in nuclear extracts is not the product of an

RNA processing activity. (A) Northern blot analysis of RNA madein nuclear extracts with pLlOl as a template. The reaction wasallowed to proceed for different times ranging from 0 min (lane 1) to60 min (lane 8). RNA was extracted at each time point and analyzedafter fractionation on a 1.5% agarose gel containing formaldehyde.After transfer to a nylon filter, the blot was hybridized to anantisense probe containingADH2 sequences and exposed to film forautoradiography. The arrow points to the most prominent transcrip-tion product. (B) Radiolabeled RNA corresponding to the ADH2sequences in pLlOl, transcribed by using RNA polymerase T7, wasincubated with nuclear extract under standard transcription condi-tions. The resulting RNA was analyzed after fractionation on a 7.5%polyacrylamide gel containing 8 M urea and exposed for autoradiog-raphy. Lane 1, size markers; lane 2, RNA prior to incubation withnuclear extract; lane 3, RNA after incubation with nuclear extract(N.E.). Markers are MspI-digested pBR322 DNA end labeled with[32p]cTp.

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YEAST POLYMERASE II TRANSCRIPTION TERMINATION 5163

with the kinetics predicted for a precursor to the major RNAspecies produced in the in vitro system. Further evidence thatthis RNA is the product of transcription and not the productof a processing reaction comes from the following experi-ment. By using T7 RNA polymerase, a sense-strand RNAwas synthesized which ends 100 nt downstream of the puta-tive termination site. This radiolabeled RNAwas incubated inthe nuclear extract under transcription conditions and ana-lyzed on a denaturing gel. It is clear from Fig. 4B that theRNA remains unchanged after exposure to the nuclear ex-tract. This result implies that the pre-RNA is not degraded inthe extract to produce the major species that we detected inthe in vitro reaction. Together, these results support thenotion that the RNA detected in vitro is a primary transcrip-tion product. Further experiments will be required to demon-strate this conclusively.A putative mRNA precursor can be detected in vivo. It is

very difficult to detect a precursor mRNA in vivo because ofrapid cleavage and polyadenylation. However, if the tran-script which we observed as a result of the in vitro transcrip-tion reaction is the bona fide precursor RNA, it is importantto demonstrate that a comparable RNA species is present inthe yeast cell. In order to address this question, we askedwhether a poly(A)- RNA approximately 100 nt longer thanthe mature RNA could be detected in RNA made from cellsharboring plasmid pLlOl.

Total RNA and poly(U)-Sephadex-fractionated RNAmade in vivo from yeast cells carrying pLlOl were subjectedto Northern blot analysis with two probes (Fig. SC): one(EcoRI) which should detect the mature ADH2 mRNA, aswell as any RNA extending beyond the ADH2 poly(A) site,and a second (AccI) which should recognize only the pre-mRNA species. The results are presented in Fig. 5A. ThisNorthern blot analysis shows two RNA species, one ofwhich is prominent and binds to poly(U)-Sephadex. Theother, less-abundant RNA does not bind to poly(U)-Sepha-dex and thus either is not polyadenylated or contains apoly(A) tail shorter than 10 nt. This RNA migrates with aslightly slower mobility than poly(A)+ RNA and in additioncontains sequence downstream of the poly(A) site (Fig. 5A,lanes 2 and 4).

In order to directly demonstrate that the higher-molecular-weight species does not contain a poly(A) tail, total RNAwas hybridized to oligo(dT) and treated with RNase H todegrade the poly(A) tail. As expected, the prominentpoly(A)+ RNA migrates faster after the RNase H treatment,reflecting the loss of the poly(A) tail (Fig. SB, lanes 1 and 2).The higher-molecular-weight species containing the down-stream sequence is unaffected by the RNase H treatment,strongly suggesting that it does not contain poly(A) residuesat its terminus (Fig. SB, lanes 3 and 4). The difference in sizebetween the two RNAs is approximately 50 nt, placing thetermination site approximately 100 nt downstream of thepoly(A) site [taking into account that the average length ofthe poly(A) tail in yeast cells is 50 nt]. This species mayrepresent the in vivo counterpart to the RNA which wedetected in the in vitro reaction.

Transcription products containing the GAL7 3' end can bepolyadenylated. It is interesting that transcription of pLlOl invitro leads to the accumulation of a putative pre-mRNAspecies. This accumulation may be due to inefficient use oftheADH2 polyadenylation site in vitro. We have establishedthat the ADH2 3'-end sequence is a very poor substrate forthe cleavage and polyadenylation reaction when added towhole-cell extracts which are competent in processing otheryeast pre-mRNAs (8a). In contrast, the GAL7 3'-end se-

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FIG. 5. Putative pre-mRNA detected in vivo. (A) Northern blotanalysis of total RNA (lanes 2 and 4) and poly(A)+ RNA (lanes 1 and3) from yeast cells carrying plasmid pLlOl. RNA was fractionatedon a 3.5% NuSieve agarose gel containing formaldehyde. Aftertransfer to nylon filters, the blots were hybridized to either theEcoRI probe (lanes 1 and 2) or the AccI probe (lanes 3 and 4). Theblot hybridized to the AccI probe was exposed for 3 days, and theblot hybridized to the EcoRI probe was exposed for 16 h. The arrowmarks the putative pre-mRNA. (B) Northern blot analysis of RNaseH treatment of pLlOl RNA. Total RNA from yeast cells carryingplasmid pLlOl was hybridized to oligo(dT). RNA was treated withRNase H (lanes 2 and 4) or not treated (lanes 1 and 3) andfractionated on a 3.5% NuSieve agarose gel containing formalde-hyde. After transfer to a nylon filter, the blots were hybridized toeither the EcoRI (lanes 1 and 2) or the AccI (lanes 3 and 4) probe.The blot hybridized to the AccI probe was exposed for 5 days andthe blot hybridized to the EcoRI probe was exposed for 16 h. Thearrow marks the putative pre-mRNA. (C) Schematic diagram show-ing the 3' end of theADH2 gene in a T7-T3 vector used to synthesizeprobes for Northern analysis. Transcription from the vector di-gested with EcoRI produces an antisense probe of 340 nt whichcontains all of the ADH2 3'-end sequence found in pLlOl. Tran-scription from the same vector digested withAccI produces a 140-ntantisense probe containing onlyADH2 sequence downstream of thepoly(A) site. The poly(A) addition sites are marked by arrows.

quence is a very efficient substrate for the RNA processingreaction in whole-cell extracts (1, 8). Therefore, we wereinterested in determining whether GAL7 transcripts werecleaved and polyadenylated in the in vitro reaction. For thisexperiment, we used plasmid pL501, which contains theGAL7 3'-end sequence in place of the ADH2 sequence.Poly(A)+ RNA synthesized from this template was selectedby poly(U)-Sephadex chromatography and subjected toNorthern blot analysis. Two different probes were used andare illustrated in Fig. 6B. One extends to the HindIll site andwill hybridize to all RNAs containing GAL7 sequence. Thesecond probe stops at the Hinfl site and will hybridize onlyto RNA containing GAL7 sequence past the poly(A) site.When the HindIII probe is used, it is clear that, in the caseof the GAL7 template, a significant amount of the in vitro-transcribed RNA indeed contains a poly(A) tail (Fig. 6A).However, the polyadenylation reaction is not as efficient as

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5164 HYMAN AND MOORE

AHindill Hinfl

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FIG. 6. GAL7 RNA made in vitro is polyadenylated. (A) TotalRNA from a standard transcription reaction mix was hybridized topoly(U)-Sephadex beads to select any RNA containing a poly(A) tailgreater than 10 nt in length. Fractionated RNA was analyzed afterelectrophoresis on 1.5% agarose-formaldehyde gels by Northernblot hybridization with antisense GAL7 probes (shown in panel B).Lanes 1 to 3 contain in vitro-transcribed RNAs. Lane 1 containsunfractionated RNA, lane 2 represents RNA which bound to thepoly(U) beads, and lane 3 contains RNA which did not bind topoly(U)-Sephadex. Lanes 4, 5, and 6 contain 5 jig of RNA fromyeast cells carrying plasmid pL501 and represent unfractionated,poly(A)+, and poly(A)- in vitro-transcribed RNA, respectively.This filter was hybridized to the HindIII probe (panel B). Lanes 7 to9 contain unfractionated, poly(A)+, and poly(A)- RNA, respec-tively, hybridized to the Hinfl probe (see panel B). The autoradio-gram for lanes 1 to 6 was exposed for 3 h, and the autoradiogram forlanes 7 to 9 was exposed for 48 h. Higher-molecular-weight RNAswere apparent after longer exposures of lanes 1 to 3. (B) Schematicrepresentation of probes for differential hybridization to GAL7RNA. The probes were made after digestion of pGAL7-1 (8) witheither HindIII (to produce an antisense transcript 266 nt in length) orHinfl (to produce an antisense transcript 100 nt in length). The Hinflsite is 7 nt downstream of the GAL7 poly(A) site (arrow).

in vivo, since much of the in vitro-transcribed RNA remainspoly(A)- (Fig. 6A, compare lanes 3 and 6). Hybridizationwith the Hinfl probe demonstrates that only the poly(A)-RNA contains downstream sequences; the poly(A)+ RNAdoes not. Even when the autoradiogram is overexposed, nopoly(A)+ RNA is observed (Fig. 6A, lanes 7 to 9). This longexposure of the autoradiogram accounts for the apparentdiscrepancy in the hybridization patterns with the two dif-ferent probes. Upon longer exposures of the autoradiogramhybridized to the HindIII probe, a similar set of RNAs aredetectable (data not shown). Together, these results indicatethat, unlike the ADH2 3' transcripts, some of the GAL7

1 2 34FIG. 7. GAL7 pre-mRNA is not cleaved or polyadenylated in

nuclear extracts. GAL7 pre-mRNA was synthesized in vitro toproduce a 266-nt transcript containing 246 nt of GAL7 sequence(lane 1). Approximately 10 nM GAL7 pre-mRNA (200,000 cpm) wasadded to a standard transcription reaction mix (lane 2), to nuclearextract under conditions (ionic strength, buffer, and temperature)favorable to cleavage and polyadenylation (lane 3), or to a yeastwhole-cell extract under standard RNA cleavage and polyadenyla-tion conditions (lane 4). The products of the reaction were analyzedon a 6% polyacrylamide-8.3 M urea gel and visualized by autora-diography. The schematic diagram to the right of the autoradiogramrepresents, from top to bottom, precursor RNA, cleaved andpolyadenylated RNA, and the 5' cleavage product.

RNA appears to be cleaved and polyadenylated, while someof the RNA is present as a putative precursor species.These results suggest that in the presence of an efficient

poly(A) site, RNA transcribed by RNA polymerase II invitro can be cleaved and polyadenylated. Perhaps this is notsurprising, since it has already been demonstrated thatpre-mRNAs can be cleaved and polyadenylated when addedto a whole-cell yeast extract (1, 7). We were interested indetermining whether the cleavage and poly(A) addition seenwith nuclear extracts was coupled to the in situ transcriptionof the RNA or, alternatively, whether exogenous pre-mRNAcould be cleaved in the in vitro transcription reaction. Toaddress this question, a radiolabeled RNA corresponding tothe GAL7 sequence present in pL501 was added to thetranscription reaction mix. Under normal transcription con-ditions, cleavage and polyadenylation of the GAL7 substratewere not observed (Fig. 7, lane 2). In order to present a morefavorable environment for the RNA processing reaction, wetested the same substrate with the nuclear extract underreagent conditions which allow cleavage and polyadenyla-tion in the whole-cell extract. We still did not detect anycleavage or polyadenylation of the substrate (Fig. 7, lane 3).We observed cleavage and polyadenylation of the GAL7substrate only when the reaction mix contained the whole-cell extract and the reaction was performed under standardpolyadenylation conditions (Fig. 7, lane 4). This result isconsistent with the idea that the cleavage and polyadenyla-tion of GAL7RNA in nuclear extracts are somehow coupledto transcription and do not occur with pretranscribed sub-strates. However, it is not clear whether such couplingoccurs in vivo.

Effect of deletion of a GAL7 poly(A) signal on transcriptionproducts in vivo and in vitro. ATA repeat sequence upstreamof the poly(A) site of some yeast genes has been shown to beimportant for cleavage and polyadenylation (1, 8, 32). Sucha TA repeat is found in the GAL7 sequence 22 nt upstream ofthe poly(A) site. A 13-nt deletion of this element preventscleavage and polyadenylation in vitro (8). In order to assessthe effects of this mutation on transcription products in vitroas well as in vivo, we constructed a derivative of plasmidpL501 which contains the GAL7 site with the deletion. RNAderived from yeast cells carrying this plasmid, pL502, was

MOL. CELL. BIOL.

YEAST POLYMERASE II TRANSCRIPTION TERMINATION 5165

A0

LO-j

0.

BCIA0

-j

c

HindTil Hinfl

CYl _ N -0 0 0 0aLa in

, I

1 2 3 4

T- Iro 0

1L

1 21 2

FIG. 8. Effect of deletion of a poly(A) signal on transcriptionproducts in vivo and in vitro. (A) RNA was prepared from yeastcells carrying plasmid pL501 (5 pLg of total RNA, lane 1) or pL502 (5p.g of total RNA, lane 2). RNAs were analyzed after electrophoresison a 1.5% agarose-formaldehyde gel by Northern blot hybridizationwith the HindIII antisense GAL7 RNA probe shown in Fig. 6B. (B)RNAs from in vitro transcription reactions with pL502 (lanes 1 and3) or pL501 (lanes 2 and 4) as a template were analyzed afterelectrophoresis on 1.5% agarose-formaldehyde gels and Northernblot hybridization with either the full-length GAL7 antisense probe(HindIII, lanes 1 and 2) or the GAL7 probe which contains onlysequences downstream of the poly(A) site (Hinfl; lanes 3 and 4).These probes are shown in Fig. 6B. (C) RNAs from in vitrotranscription reactions with pLlOl (lane 1) or pL401 (lane 2) as thetemplate were analyzed after electrophoresis on a 1.5% agarose-

formaldehyde gel and Northern blot hybridization with an ADH2antisense probe.

analyzed by Northern blot hybridization. The pL502 tem-plate does not support proper 3'-end formation at the GAL7site in vivo (Fig. 8A, lane 2). In the pL502 plasmid,readthrough of the 3'-end sequence would lead to subse-quent splicing of the ribosomal protein 51 intron and produc-tion of 0-galactosidase (see Fig. 1A). As expected, the levelsof 3-galactosidase produced from the pL502 template are

equal to the levels produced from a template containing a

nonfunctionalADH2 poly(A) site (data not shown) (15). Thisdemonstrates that the pL502 RNA is stable and does indeedcontain sequences which extend into the lacZ gene.

Hybridization of the pL501 and pL502 in vitro-transcribedproducts with the differential probes (Fig. 6B) is shown inFig. 8B. The fastest-migrating product of plasmid pL501shows no hybridization to the downstream probe and mostlikely corresponds to mature poly(A)+ RNA (Fig. 8B, com-

pare lanes 2 and 4). Because the two higher-molecular-weight bands hybridize to both probes, they contain down-stream sequences and probably represent uncleaved RNAs.In the case of the GAL7 template with the TA deletion, thesize of the shorter band suggests that it extends only a shortdistance past the GAL7 poly(A) site (Fig. 8B, lanes 1 and 3).These results show that the mutation prevents cleavage andpolyadenylation of the pL502 GAL7 substrate in vitro as wellas in vivo but does not affect the transcription termination or

pausing which we observed in vitro just downstream of theGAL7 poly(A) site.We previously described point mutations in the ADH2 3'

end which resulted in readthrough transcription of theADH2sequence in vivo (16). One of these mutations, carried bypL401, is a T to G transition 75 nt upstream of the ADH2poly(A) site. We were unable to assess the effects of thismutation on cleavage and polyadenylation because, as notedabove, theADH2 sequence is a very poor substrate for these

reactions in vitro. When plasmid pL401 is used as a templatefor the in vitro transcription reaction, we observe RNAswhich contain sequences beyond the ADH2 poly(A) site thatappear identical to the RNA synthesized from the wild-typeplasmid (Fig. 8C). Therefore, it is likely that this pointmutation is affecting not termination but rather RNA pro-cessing, perhaps in a manner comparable to the TA deletionof the GAL7 sequence discussed above.

DISCUSSION

In this report, we demonstrate the use of the yeastcell-free system originally developed to study initiation oftranscription for examining transcription termination. Westudied two yeast sequences, one from the region surround-ing the poly(A) site of the ADH2 gene and the other from theequivalent region of the GAL7 gene. In both cases, RNAproducts with 3' ends within the test sequences were ob-served only in the presence of the appropriate DNA templateand in the presence of nucleoside triphosphates. In addition,the transcripts synthesized in vitro were dependent on theactivity of RNA polymerase II. While both of the 3'-endregions tested are very efficient at directing polyadenylationin vivo, poly(A)+ RNA was observed only for the GAL7template. Interestingly, in vitro transcription from both theADH2 and the GAL7 templates produced a discrete speciesof poly(A)- RNAs which extended a short distance down-stream of the in vivo poly(A) site. In the case ofADH2, theends of these transcripts are in a region approximately 100 ntpast the poly(A) site. Importantly, we have detected apoly(A)- RNA in vivo which also extends approximately100 nt downstream of the ADH2 poly(A) addition site. Thepresence of this RNA suggests that a precursor species canbe detected in vivo but, as expected, is present at very lowconcentrations in the cell.The most plausible explanation for the presence of the

short poly(A)- transcripts made as a result of transcriptionin vitro is that they represent the primary transcriptionproduct, that is, the RNA present prior to cleavage andpolyadenylation. Indeed, the emerging picture of 3'-endformation of yeast mRNAs requires that RNA polymerase IIterminate transcription immediately downstream of thepoly(A) site. This is based on at least three lines of evidence.First, it has been shown that transcription through either anautonomous replication sequence (ARS) or a centromere(CEN) destabilizes plasmids. However, when sequencescontaining yeast poly(A) sites are cloned just upstream ofthese elements, the plasmids are stabilized (33, 37), suggest-ing that transcription no longer proceeds into the ARS orCEN. Osborne and Guarente used a different assay fortranscription termination, one which measured DNA topol-ogy in actively transcribed regions versus untranscribedregions (25). Insertion of the CYCl 3'-end sequences into aplasmid resulted in an increase in the negative supercoilingof the DNA, consistent with the idea that these CYCIsequences cause transcription termination close to the pointwhere the sequence was inserted in the plasmid. Finally, ithas been shown that the insertion of poly(A) sites into theintrons of two different yeast transcripts resulted not inspliced products but in truncated RNAs mapping to theinserted sequences (15, 17, 30). This result was puzzling,given the observation that insertion of a poly(A) site into anintron of a mammalian gene results in splicing of the intronrather than cleavage and polyadenylation (2, 6, 21). How-ever, if these yeast sequences are directing immediate tran-scription termination as well as polyadenylation, the 3'

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splice site would not be synthesized, and any competitionbetween the splicing and cleavage-polyadenylation of thepre-mRNA would be eliminated. Termination often occurs1,000 nt or more downstream of the poly(A) site in mamma-lian genes (28), and this difference in the position of termi-nation could explain the discrepancy between yeast andmammalian genes.Russo and Sherman point out that a possible reason for

this difference between S. cerevisiae and higher eukaryoteslies in the differences in genome organization; yeast genestend to be spaced close together (33). Recent experiments byAshfield et al. (3) have shown that a polymerase pause site islocated just downstream of the poly(A) site of the humancomplement gene C2, which is only 421 nt upstream of thefactor B transcription start site. This provides further evi-dence that even in mammals, it is important to provide asignal for transcription termination close to the 3' end of agene in order to avoid transcription interference when genesare closely spaced. As discussed in this report, we haveobserved synthesis of poly(A)- RNAs which end just down-stream of the poly(A) site. In addition, similar results haverecently been reported in a study of transcription termina-tion of the CYCl gene (41). These results are fully consistentwith the predictions of the in vivo studies of yeast mRNA3'-end formation.We have not yet determined whether the poly(A)- tran-

scripts that we detected in vitro are the products of tran-scription termination and release of the nascent transcriptfrom the ternary complex or the products of RNA poly-merase II pausing. Because pre-mRNA added to the reactionmix is not processed, it is unlikely that the products that wedetected in vitro are the result of a nuclease activity whichspecifically degrades the RNA to a site just downstream ofthe poly(A). We favor the model that 3'-end formation ofeukaryotic mRNAs involves polymerase pausing at specificsequences downstream of yeast poly(A) sites. Although wedo not have direct proof of this model, this conclusion isbased on the observation that a mutation in the GAL7 orADH2 poly(A) signal results in complete readthrough oftranscription in vivo. However, transcription in vitro withthe same templates resulted in the accumulation of pre-mRNAs. This apparent contradiction between the in vitroand in vivo results can be resolved if what we are detectingin the in vitro reaction are the products of RNA polymerasepausing at specific sites downstream of the poly(A) site. Ourhypothesis is that this pause is required to search for anupstream poly(A) site. If a poly(A) site is recognized,perhaps by formation of a processing complex or as aconsequence of processing, polymerase dissociates from thetemplate. In the case of the mutated poly(A) site, polyade-nylation does not occur and the polymerase eventuallymoves on. Thus, the simplest interpretation of the data leadsus to propose a mechanism for 3'-end formation whichinvolves two signals, one for cleavage and polyadenylationand another for RNA polymerase pausing. Since we did notdetect any poly(A)+ RNA for ADH2 in vitro, an attractiveextension of the model is that weak poly(A) sites mayrequire strong pause sites for efficient 3'-end formation.Together, these results support the hypothesis, first sug-gested by Proudfoot (28), that the signal for transcriptiontermination consists of a poly(A) site coupled with a termi-nation sequence or pause site. Fractionation of the nuclearextracts, including demonstration of a ternary complex atputative pause sites, will clarify this model. These andsimilar experiments are under way.

Studies on purified mammalian RNA polymerase II have

revealed sequences which cause transcription termination invitro (19). These intrinsic termination sites generally consistof two closely spaced runs of T residues on the nontran-scribed strand. This termination signal can cause DNAbending, and it has been suggested that this structuralchange may trigger termination. The sequences that we havestudied do not contain such T stretches but may containother sequences which cause DNA bending. Some mamma-lian transcription termination-pause sites have been definedby nuclear run-on analysis (12). There is no apparent se-quence homology between these termination sequences, butthey appear to be adenosine rich on the nontemplate strand.Interestingly, the GAL7 sequence described in our studydoes indeed contain a long stretch of adenosine residues(A14GA6GA2) 50 nt downstream of the poly(A) site, in aregion corresponding to the prominent in vitro termination-pause sites. On the other hand, the relevantADH2 sequenceis not A rich but rather C rich. We have previously shownthat sequences between 17 and 54 nt downstream of theADH2 poly(A) site are required to direct 3'-end formation invivo (15). One of the prominent poly(A)- transcripts whichwe detected as a result of transcription in vitro also maps toapproximately this position, while the others fall in a regionfurther downstream which we have not yet analyzed indetail.

Interestingly, we observed cleavage and polyadenylationof the GAL7 RNA only when its synthesis was coupled totranscription in a nuclear extract. Exogenous GAL7 RNAwas not used as a substrate for cleavage and polyadenylationeven though the poly(A) site was present. This is in contrastto GAL7 RNA in yeast whole-cell extracts, in which it is anexcellent substrate for cleavage and polyadenylation (1, 7,8). We do not know what causes the difference betweenthese two extracts, but we presume that whatever contrib-utes to the coupling of transcription and cleavage-polyade-nylation has been lost in the preparation of the whole-cellextracts. We note that it is formally possible that thepoly(A)+ RNA that we observed is the product of transcrip-tion termination and end polyadenylation, without endonu-cleolytic cleavage. However, we consider this unlikely be-cause of the efficiency of cleavage and polyadenylation ofthe GAL7 substrate in yeast whole-cell extracts.The application of the yeast in vitro transcription system

to a study of transcription termination should enable us todefine the precise sequence requirements for polymerasepausing and/or termination, which in turn may help to definethe actual mechanism for transcription termination. We havenot yet been able to establish whether the signals requiredfor the termination that we detected in vitro are present inthe RNA or the template, are dependent on features such asDNA bending or RNA secondary structure, or are the resultof the binding of proteins acting as road blocks to RNApolymerase. These are questions which deserve furtherstudy.The data presented in this report support the idea that

3'-end formation in S. cerevisiae and that in higher eukary-otes proceed by comparable mechanisms. In both systems, afunctional polyadenylation signal is required for transcrip-tion termination in vivo, and in both systems this appears tobe mediated by RNA polymerase pausing just downstreamof the RNA cleavage site. To date, no trans-acting factorswhich are required for transcription termination have beenidentified. However, with the yeast in vitro system coupledwith genetic analysis, we hope to learn more about themechanism of this reaction and about the other componentsinvolved in transcription termination.

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ACKNOWLEDGMENTS

We are very grateful to Shelley Berger for providing us with manyreagents as well as for numerous helpful suggestions. We thank StevenSchneider and Sam Clark for excellent technical assistance and JieChen for providing us with pGAL7-1, pGAL7-3, GAL7 pre-RNA, andyeast whole-cell extracts. We thank all the members of our laboratoryfor fruitful discussions and help with the manuscript. We are grateful toShelley Berger, Dean Dawson, Michele Flatters, Anne Skvorak, andAndrew Wright for critically reading the manuscript.

This work was supported by NIH grant GM42760 to C.L.M. andNIH grant GM12818 to L.E.H.

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