Proc. Natl. Acad. Sci. USAVol. 84, pp. 8306-8310, December 1987Biochemistry

A poly(A) addition site and a downstream termination region arerequired for efficient cessation of transcription by RNApolymerase II in the mouse IBmaJ-globin gene

(transcription rate analysis/site-directed mutations)

JOHN LOGAN*, ERIK FALCK-PEDERSENt, JAMES E. DARNELL, JR.t, AND THOMAS SHENK§*Agricultural Research Division, American Cyanamid Company, Princeton, NJ 08540; tDepartment of Microbiology, Cornell University Medical College, NewYork, NY 10021; tRockefeller University, New York, NY 10021; and §Department of Molecular Biology, Princeton University, Princeton, NJ 08544

Contributed by James E. Darnell, Jr., August 11, 1987

ABSTRACT Sequence elements within the mouse P1"j-globin transcription unit required for efficient termination oftranscription by RNA polymerase II have been delineated. Tofacilitate nascent-chain analysis of termination, the DNAsegment in which transcription ceases was introduced into theadenovirus chromosome within its ElA transcription unit. Two13-globin DNA elements were required to effect efficient ter-mination: an upstream sequence that includes two poly(A)addition signals and a downstream region previously shown tobe where RNA synthesis stops. The role of poly(A) addition intermination was established by introduction of several singlebase pair substitutions into the AATAAA polyadenylylationmotifs. These mutations inhibited both polyadenylylation andtermination within the P-globin DNA segment. Therefore,poly(A) addition appears to be a prerequisite for efficienttermination.

It is well established for a variety of transcription units inhigher eukaryotes that RNA polymerase II does not termi-nate at the 3' end ofmRNAs. This was first demonstrated forthe adenovirus major late unit (1, 2) and subsequently wasextended to a variety of cellular genes (3-8). The polymerasetranscribes past the site of the 3' end that is later generatedby endonucleolytic cleavage of the primary transcript. Thiscleavage is followed by poly(A) addition for most mRNAs(for reviews, see refs. 9 and 10). Termination regions havebeen mapped for several genes well downstream of theirpoly(A) addition sites [e.g., mouse 83-globin (3), mousea-amylase (7), chicken ovalbumin (11) (for review, see ref.10)]. The mouse praJ-globin gene (12) is perhaps the beststudied. Termination has been localized to a region 600-1500nucleotides (nt) downstream of the poly(A) site by analysis ofnascent RNA labeled both in isolated nuclei (3, 13-15) andwhole cells (15).We have previously shown that the mouse pmah-globin

terminator domain can function when incorporated into theadenovirus chromosome within a region encoding the 3'exons of ElA mRNAs (16). The high adenovirus templatecopy number after infection and the resulting high levels ofRNA synthesis within infected cells facilitated nascent-chainanalysis of transcriptional termination. Insertion of a DNAsegment spanning the region in which the decrease in ,3-globin-specific transcription rate occurs had no effect ontranscription through the ElA 3' exon. A larger segment,however, including an additional 800 base pairs (bp) upstreamof the region in which transcription normally stops, signalledtermination within the correct region. The larger insertincluded the two poly(A) addition motifs that follow the,B-globin coding region, leading us to speculate that termina-

tion might first require cleavage and polyadenylylation of thenascent transcription.We have now tested this hypothesis. Deletion analysis of

the 3-globin insert on the adenovirus chromosome demon-strated that two domains are required for termination: adownstream region in which transcription stops and anupstream region including the poly(A) addition sites. TheAATAAA motifs that signal polyadenylylation were mutatedby the introduction of several base pair substitutions. Re-sulting variants failed to utilize the altered P-globin poly-adenylylation signals and displayed a markedly reducedefficiency of termination. Thus, polyadenylylation is a pre-requisite for efficient termination by RNA polymerase II atthe mouse Pmai-globin termination domain.

MATERIALS AND METHODS

Plasmids, Viruses, and Cells. Mouse frnaJ-globin DNAsequences were from the genomic insert in Xgtwes'MfG2(12). A segment designated gDEF extending from the Bal Isite (1233 bp from the cap site) to the Bgl II site (2790 bp fromthe cap site) was inserted into the Xba I cleavage site ofpMLP6 (17), as previously described (16). Derivatives of thegDEF insert were prepared by excising segments usingconvenient restriction endonucleases [gF (16); gD, gEF, andgDF (Fig. 1)]; by inverting segments [gD-E-F- (16);gD+F- (Fig. 1)]; or by introducing point mutations [gD"EF(Fig. 1)]. Point mutagenesis used oligonucleotides carryingthe mutant sequence to prime second-strand synthesis of agD fragment subclone carried in an M13 recombinant, es-sentially according to the procedure of Zoller and Smith (18).Mutant progeny were identified by differential hybridizationusing the mutant oligonucleotide as probe DNA, and thealteration in positive clones was confirmed byDNA sequenceanalysis. The procedure was done twice sequentially to alterboth AATAAA sequences [the 5' site is the normal poly(A)addition site for f3-globin] contained within the D segment.The mutated segment was then inserted into pMLP6 as partof a gDEF insert and termed gD"EF (D" designates the twosets of point mutations within the D segment; Fig. 1).

Viruses were reconstructed by the overlap recombinationmethod (19) using pMLP6 derivatives linearized with EcoRI(cleaves at the joint between plasmid DNA and the left-endterminus of the adenovirus insert) and the 3.8-100 map unitsegment ofthe adenovirus type 5 (AdS) variant ofdl309 strain(20) generated by cleavage with Xba I. The parent to the,B-globin insert-containing viruses is strain sub360-LO(termed sub360 in this report), which has been described (17).

Abbreviations: pfu, plaque-forming units; gD, gE, gF, etc., segments"D," "E," "F," etc., of the plrna-globin gene and various combi-nations including inverted segments (-) and two sets of pointmutations (") as indicated in Fig. 1; Ad5, adenovirus type 5; sub360;virus strain sub360-LO.

8306

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Proc. Natl. Acad. Sci. USA 84 (1987) 8307

lal Ia2 Ia3 EIA PROBES

12S EIA2S - mRNAs sub360

II Xbaa ll500 bpJMLTCR -

-5EF ~1 B-GLOBINI- Isub 360-gF

I I I I sub360-gDEF11 14 4 I sub360-gD-E-F-

sub360-g DI I I sub360-gEF

IF-H| I sub360-gDF4-l I sub360-gD+F-

F- -11 Isub360-gD"EF. POLY(A) SITES

rEGGAAATAAATGA.._ GGAAACCAGGGA*0 00

L.-.ACAAATAAAAAG--- ACCAGGAAAAAG0 0*

FIG. 1. Diagrammatic representations of the Ad5 ElA transcrip-tion unit, the alteration present in sub360 and sub360 derivatives thatcontain segments of the I31'ai-globin gene. (Top) Location of ElA-specific probe DNAs (lal, a2, and 1a3) (see Materials and Meth-ods), the ElA mRNAs with introns indicated by spaces (13S, 12S,and 9S), and sub360 with 241 bp from the major late-transcriptionalcontrol region (MLTCR) substituted for a portion of the ElA controlregion in sub360. (Middle) sub360 derivatives that contain all orportions of the 8mai-globin DEF segment (bp 1233-2790 relative tothe globin cap site) inserted at the Xba I cleavage site within the 3'exons of ElA mRNAs. Portions of the gDEF region present in eachconstruct are represented by bars, and left-facing arrowheads des-ignate segments the orientation of which is reversed as compared totheir normal orientation within the /8-globin transcription unit.(Bottom) Base pair substitutions (large dots) introduced to alter thetwo poly(A) addition motifs within the gD fragment.

Strain sub360 is a phenotypically wild-type virus that con-tains pMLP6 sequences to the left of its Xba I cleavage site(at 3.8 map unit) and strain d1309 sequences to the right of theXba I site. The major late-transcriptional control regionreplaces the ElA control region in this virus and its 13-globininsert-containing derivatives, facilitating high-level expres-sion of the ElA unit late after infection. ElA expression forsub360 is indistinguishable from wild-type viruses early afterinfection.

Viruses were propagated on human 293 cells that containand express the left-end 11% of the AdS chromosome (21).Transcription experiments were performed in infected HeLaspinner culture cells. Cell line 293 and HeLa cells were bothpropagated in medium containing 10% calf serum.

Preparation and Analysis of RNA. HeLa cell cultures wereharvested 4 hr after infection at a multiplicity of 25 plaque-forming units (pfu) per cell. For RNA blot analysis, cyto-plasmic RNA was prepared as previously described (22), andpoly(A)+ RNA was selected by chromatography on oligo-(dT)-cellulose. Electrophoresis, transfer to nitrocellulose,and hybridization were as described (23). To measure tran-scription rates, nuclei were isolated by Dounce homogeni-zation (24), incubated for 10 min at 30°C in the presence of[32P]UTP (750 ,uCi/ml, 410 Ci/mmol; 1 Ci = 37 GBq), nuclearRNA was isolated (22), degraded by treatment with 0.2 MNaOH for 10 min at 0°C, and hybridized to single-strandedprobe DNA bound in dot or slot format to nitrocellulosefilters (25). Each dot or slot contained 5 ,ug of denaturedDNA. After hybridization, filters were washed, digested withribonuclease A, and radioactivity was detected by exposureto preflashed Kodak XAR-7 film. Resulting exposures werequantitated with an LKB densitometer that scanned theentire dot or slot. Probe DNAs were as follows: plal, pla2,

and pla3 contain Ad5 ElA-specific segments between se-quence position 1-1010, 1010-1336, and 1336-1674, respec-tively; pibS' and plb3' contain AdS ElB-specific segmentsbetween position 2047-2501 and 3327-3823, respectively; p2acontains an AdS E2A segment located between 63.6-68.0map units; pgD, pgE, and pgF contain f-globin-specificsegments located at positions 1233-1542, 1542-1985, and1985-2790, relative to the 13-globin cap site respectively; p,8-tcontains a human ,8-tubulin insert (26) and p,/-actin containsa segment of the human 8-actin gene (27).

RESULTS

Two Sequence Elements Are Required for Efficient Termi-nation. It is difficult to study the formation of nascent RNAfrom individual transcription units residing within cells orfrom recombinant plasmids subsequent to transfection due totheir relatively low levels of transcriptional activity. Incontrast, it is relatively easy to monitor the formation ofnascent RNA chains arising from individual adenovirustranscription units because many copies of the viral chromo-some are present and transcriptionally active within infectedcells. Therefore, a series of AdS sub360 derivatives wereconstructed to study the mouse p-ma1-globin terminationregion. Globin-specific DNA segments were inserted at anXba I cleavage site within the region encoding the 3' exon ofviral ElA mRNAs (Fig. 1). The ability of the globin segmentsto direct termination could then be assayed by monitoringtranscription rates within the ElA transcription unit up-stream and downstream of the insert.

Previously, we determined that insertion of the ,8-globin Ffragment (gF) that contained the sites at which transcriptionterminates within the 83-globin unit had no effect on ElAtranscription, whereas the entire gDEF segment signalledtermination (16). Further, the gDEF segment had to beinserted in its normal orientation. When it was reversed(gD-E-F-, Fig. 1), it did not function (16).To identify sequences within gDEF necessary for termi-

nation, additional sub360 derivatives were prepared contain-ing gD, gEF, or gDF inserts (Fig. 1). Termination wasquantitated as before (16) by hybridization of labeled nascentRNA prepared from isolated nuclei to DNA segments up-stream of the insert (probes lal and 1a2; Fig. 1), across theinsert (probes gD, gE, gF; Fig. 1), and downstream of theinsert (probe 1a3; Fig. 1). The resulting dot hybridizationautoradiograms are displayed in Fig. 2A; key quantitativedata are listed in Table 1, Experiment 1, and the percenttermination induced by each insert [l-(1a3 OD/1a2 OD) x100] is diagrammed in Fig. 2B.A low level of termination was consistently observed for

sub360 that carried no insert (Fig. 2B). Presumably thisrepresents the frequency at which the polymerase fails tosuccessfully traverse the entire 1a2 and 1a3 regions undernormal conditions (28). Insertion of gDEF increased thefrequency of termination by a factor of 10. The percentage oftermination measured for the gDEF insert varied from about83% to 96% in different experiments. The gD and gEFsegments each directed termination at reduced levels, 18%and 37%, respectively. The gDF insert directed terminationat a frequency of 73%, approaching the range of the entiregDEF region. Further, the gF fragment could be inverted,and the insert continued to function (gD+F-, 75% termina-tion), indicating the signal within the F region is eitherorientation independent or reiterated on each strand.Although each inserted segment induced enhanced levels

of termination as compared with the sub360 parent, it is clearthat the gE fragment can be deleted from the gDEF segmentiwith only a modest reduction in termination. Further, be-cause neither the gD (Fig. 2B) nor gF fragment (16) alone

Biochemistry: Logan et al.

Proc. Natl. Acad. Sci. USA 84 (1987)

A0@

0[jai a2gD 0

mgEgFto3 sub360Ib5'Ib3 pBR *-

2a-t BL 0 * r1

0*gEF

B*000

@0gDEF

000*

...0':* 0gDF

... 0@ 0gD

0* .I0

*0gD+F-

lal la30 0

U- (D g- rw0 uz .M0c

gF0

LL (D

LL = .0

oC C tUo

PROBE

16hrEXPOSURE

0 - * - S -EIA(gDEF)

-gF

EIA(sub360)gD

FIG. 2. Transcription rate analysis for determination of termina-tion frequencies. (A) Nuclei were prepared from HeLa cells at 4 hrafter infection at a multiplicity of 25 pfu per cell with sub360 or itsderivatives. Nascent 32P-labeled RNA synthesized during elongationin isolated nuclei was hybridized to a nitrocellulose filter thatcontained plasmid DNAs (5 ,ug/dot) as indicated in box. Probes: lal,1a2, and 1a3 (AdS EMA); gD, gE, and gF (f-globin); lbS' and 1b3'(AdS E1B); pBR (pBR322); 2a (AdS E2A); ,8-t (human ,8-tubulin); BL(blank). (B) Bar graph depicting the percent of termination across the/3-globin insert [1 - (1a3/1a2) x 100] for each insert. Calculationswere based on the quantitative data in Table 1.

signals efficient termination, each must contribute a se-quence element essential for the process.

Poly(A) Addition Is Required for Efficient Termination. ThegD fragment contains two AATAAA motifs, and the up-stream copy functions as the normal poly(A) addition site forthe Prma-globin transcription unit (29). We previously dem-onstrated that this polyadenylylation site is utilized in sub360-gDEF (16), and it seemed possible that the contribution ofgDto termination might involve the polyadenylylation reaction.Therefore, site-directed mutagenesis was employed to intro-duce base pair substitutions into both AATAAA motifswithin the gD segment (Fig. 1). The virus carrying thismutated gD region is termed sub360-gD"EF. RNA blotanalysis was used to identify the sites at which mRNAsencoded by this variant were polyadenylylated (Fig. 3).Several different probe DNAs (lal, 1a3, and gF; see Fig. 1)were used to allow positive identification of mRNAs. Cellsinfected with sub360-gD"EF contained drastically reducedquantities of cytoplasmic mRNA polyadenylylated at theP-globin site (gD). Rather, the ElA-specific mRNAs were

Table 1. Efficiency with which segments of the 6-globintermination domain direct cessation of transcription

Probes, OD units

Virus 1a2 1a3 1a3/1a2 Termination, %

Experiment 1sub360 3.21 2.91 0.91 9sub360-gDEF 3.15 0.37 0.12 88sub360-gD 1.31 1.07 0.82 18sub360-gEF 2.96 1.85 0.63 37sub360-gD+F+ 2.54 0.69 0.27 73sub360-gD+F- 1.31 0.33 0.25 75

Experiment 2sub360 1.11 1.04 0.94 6sub360-gDEF 0.87 0.04 0.05 95sub360-gD"EF 1.03 0.70 0.68 32

The relative optical densities ofdots shown in Fig. 2 or slots shownin Fig. 4 were determined using an LKB densitometer and software.Background from dots or slots containing pBR322 DNA was sub-tracted from 1a2 and 1a3 measurements. The 1a2 and 1a3 probeDNAs were 326 and 334 bp, respectively, so it was not necessary tocorrect optical densities for differences in probe length.

_S.

f

_. ...... ..

: ,*

160 hrEXPOSURE-gF

-gD

FIG. 3. RNA blot analysis of ElA-specific mRNAs present inHeLa cells infected with sub360 and its derivatives. Cytoplasmicpoly(A)+ RNA was prepared 4 hr after infection at a multiplicity of25 pfu per cell. Blot analysis utilized either lal, 1a3, or gF probeDNAs. X-ray film was exposed to the resulting nitrocellulose sheetsfor either 16 hr (Upper) or 160 hr (Lower). The location ofpolyadenylylation sites used to produce various ElA-specificmRNAs are designated [ElA (gDEF), gF, EMA (sub360), and gD].Utilization of the normal ElA-polyadenylylation site gives rise to asmaller mRNA in the case of sub360 [mRNA is designated EMA(sub360)] than for sub360-gD'EF that carries an insert within the EMAcoding region [mRNA is designated EMA (gDEF)].

polyadenylylated at the normal ElA site and to a lesser extentat an AATAAA site within gF (15).

Next, the ability of gDEF to induce termination wasassayed. Autoradiograms of slot blots are displayed in Fig.4A; key data are tabulated in Table 1, Experiment 2, and thepercent termination induced by inserts is diagrammed in Fig.4B. In this experiment the parental virus, sub360, displayed

A B80j

sub360 gDEF- lal amiit lal

IaF *.FE -.DED Dm1a3 1a3actinr pBRpBR pBR

gD EF- lal- Ia2_m F_: E

D-1a3pBRpBR

z0

z

24O

zwL)20w0w

I0(D.0U'

ioLL cs.

FIG. 4. Transcription rate analysis for determination of termina-tion frequencies. (A) Nuclei were prepared from HeLa cells at 4 hrafter infection at a multiplicity of 25 pfu per cell with sub360 or itsderivatives. Nascent 32P-labeled RNA synthesized during elongationin isolated nuclei was hybridized to a nitrocellulose filter thatcontained plasmid DNAs (5 ,ug/slot). Probes: lal, 1a2, 1a3 (AdSElA); gD, gE, and gF (J3-globin) and pBR (pBR322). (B) Bar graphdepicting the percent of termination across the /8-globin insert [1 -(1a3/1a2) x 100] for each variant. Calculations were based on thequantitative data in Table 1.

i

8308 Biochemistry: Logan et al.

Proc. Natl. Acad. Sci. USA 84 (1987) 8309

a 6% termination frequency, whereas sub360-gDEF termi-nated at 96% efficiency. The sub360-gD"EF polyadenylyla-tion variant exhibited a substantially reduced terminationfrequency, 32%.We conclude that polyadenylylation within the gD region

is required for efficient termination within the gF region. Theresidual termination observed for sub360-gD"EF could resultfrom the low level of polyadenylylation observed within thegD region and at the AATAAA sites near the 5' boundary ofthe gF fragment (Fig. 3, Lower).

DISCUSSIONThe main conclusion of this work is that two sequenceelements are required for efficient termination by RNApolymerase II within the ,8cJ-globin gDEF region. The firstelement is an AATAAA polyadenylylation motif locatedwithin the gD region, and the second element is comprised ofstop sites located several hundred base pairs downstream inthe gF region (Figs. 2 and 3 and Table 1). Each sequencealone has only a slight effect on termination; both arerequired for efficient cessation of transcription.These results lend strong support to our earlier speculation

that poly(A) addition triggers termination (16, 30). As yet itis not clear how general this observation will prove to be,although a similar conclusion has been reached recently byWhitelaw and Proudfpot (31) for the human a-globin gene.However, Baek et al. (32) and Sato et al. (33) have describeda termination site downstream of the human gastrin gene thatsignals polymerase II termination in plasmid constructs thatdo not contain an upstream poly(A) addition site. Termina-tion measurements in this case were indirect and did notutilize nascent-chain analysis. Nevertheless, the human gas-trin gene may contain a very strong element equivalent to thedownstream gF region that can function without priorpoly(A) addition.

It would make good sense for polyadenylylation to be ageneral prerequisite for RNA polymerase II terminationbecause this would assure that mRNA-coding sequenceshave been completely transcribed before a termination eventoccurred. A number of transcription units that exceed 100kilobases in length are known, and even infrequent randomtermination before an effective poly(A) site could substan-tially decrease mRNA formation in such cases. Such acoupling would play a key role in transcription units thatutilize alternative poly(A) addition sites to regulate geneexpression. One example would be the switch from produc-tion of membrane-bound IgM to secreted IgM, which in-volves the use of different polyadenylylation sites (34, 35)with a decrease in downstream transcription when the firstpoly(A) site is utilized (36).

./RNA

I I IllisIiTATA CAP PREMATURE

TERMINATION

What is the mechanism through which polyadenylylationand termination are coupled? Perhaps, cleavage of thenascent transcript at the poly(A) addition site, which canoccur before the polymerase has completed transcription ofthe unit (1), alters the conformation of the nascent RNA insuch a way that the transcription complex is destabilized andterminates at the next gF-like site it encounters. The cleavageevent generates an uncapped 5' end that could be rapidlydegraded, again possibly destabilizing the transcription com-plex. Alternatively, the polymerase complex might carry anantitermination factor with it (Fig. 5). The factor could leavethe complex at the polyadenylylation site, possibly markingthe nascent RNA for cleavage and simultaneously altering thetranscription complex, preparing it to terminate at the nextappropriate site. Polyadenylylation might not be strictlycoupled to transcription because cell-free extracts can per-form the cleavage and poly(A) addition reactions in theabsence of active transcription (37, 38). Nevertheless, themodel fits well with the observation that polyadenylylationactivity cosediments with adenovirus transcription complex-es (39). Similar models have been proposed to explain theobservation that herpes simplex virus thymidine kinase orsimian virus 40 late mRNAs synthesized from an RNApolymerase I promoter fail to be polyadenylylated (40), andto rationalize the dependence of small nuclear RNA 3' endformation on a small nuclear RNA transcriptional controlregion (41, 42). The antiterminator proposed here is reminis-cent of the phage X Q protein (43). This factor recognizes theE. coli RNA polymerase at the pR' promoter and enables itto read through the tR' termination site (44).

It is clear, however, that RNA polymerase II terminationis not always coupled to a. polyadenylylation event. Prema-ture termination within a few hundred bases ofthe RNA startsite is common. In Chinese hamster cells about one-half of allpolymerase II initiation events end in premature termination(24). Once RNA chains reach a length of 1000-2000 nt,termination seems not to occur until a poly(A) site has beencrossed (45). These early results suggested that two types ofpolymerase complex might exit-those competent to go tocompletion and those destined to terminate early. Furtherevidence for this notion comes from recent experiments onthe S2 elongation factor (46, 47). This factor enables RNApolymerase II to read past premature termination sites withinthe adenovirus major late-transcription unit. Antiterminationfactors may be required generally for successful elongation,and overcoming premature termination may be an importantregulatory event in transcription. The MYC gene is a case inpoint (48, 49). In growing HL60 human promyelocytic cellsit is transcribed completely about 30% ofthe time and abortedabout 70% of the time. When the cells are treated with

+RNA

/.IAATAAA TERMINATION

REGION

FIG. 5. Model for the mechanism coupling poly(A) addition and termination. Double line at bottom of the diagram represents the DNAtemplate on which various landmarks are'designated. RNA polymerase II transcription complex (II) is represented by a circle, presumptiveantiterminator factor (AT) by a rectangle, and RNAs by wavy lines. The model proposes that two classes of transcription complex are directedto start sites (CAP) by upstream regulatory elements (including the TATA motif). The two classes differ by the presence or absence of anantiterminator (AT) factor. Complexes lacking the AT factor cease transcription at premature termination sites. Complexes including the ATfactor read through premature stop sites, but such complexes lose the factor at a poly(A) addition site (AATAAA) and then cease transcriptionat the next termination region.

Biochemistry: Logan et al.

Proc. Natl. Acad. Sci. USA 84 (1987)

retinoic acid inducing differentiation and cessation ofgrowth,over 90% of MYC transcripts terminate prematurely.

It is possible that premature termination events and thecessation of transcription downstream of a poly(A) additionsite are unrelated and proceed by different mechanisms.However, the two events could be closely related. The regionin which termination occurs downstream of a poly(A) sitemight be functionally analogous to a premature terminationsite. Both termination events occur when the nascent RNAchain is relatively short, either due to proximity to theinitiation site or polyadenylylation cleavage site. If, assuggested above, a short nascent chain somehow destabilizesthe transcription complex, making it sensitive to a termina-tion signal, then the two termination events could proceedthrough the same mechanism. The model postulating anantitermination factor can also easily accommodate prema-ture termination events (Fig. 5). Premature terminationswould result from initiation by transcription complexeslacking the antitermination factor. Complexes containing thefactor would not become susceptible to termination signalsuntil the antitermination factor left the complex at a poly(A)addition site.

This work was supported by grants from the National Institutes ofHealth (CA 38965) and American Cancer Society (NP571 andMV271P). T.S. is an American Cancer Society Research Professor.

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