mechanism of poly(a) signal transduction to rna polymerase ii in vitro

MOLECULAR AND CELLULAR BIOLOGY,0270-7306/01/$04.00�0 DOI: 10.1128/MCB.21.21.7495–7508.2001

Nov. 2001, p. 7495–7508 Vol. 21, No. 21

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Mechanism of Poly(A) Signal Transduction toRNA Polymerase II In Vitro

DONG P. TRAN, STEVEN J. KIM, NOH JIN PARK, TIFFANY M. JEW, AND HAROLD G. MARTINSON*

Department of Chemistry and Biochemistry, University of California at Los Angeles,Los Angeles, California 90095-1569

Received 29 March 2001/Returned for modification 25 May 2001/Accepted 26 July 2001

Termination of transcription by RNA polymerase II usually requires the presence of a functional poly(A)site. How the poly(A) site signals its presence to the polymerase is unknown. All models assume that the signalis generated after the poly(A) site has been extruded from the polymerase, but this has never been testedexperimentally. It is also widely accepted that a “pause” element in the DNA stops the polymerase and thatcleavage at the poly(A) site then signals termination. These ideas also have never been tested. The lack of anydirect tests of the poly(A) signaling mechanism reflects a lack of success in reproducing the poly(A) signalingphenomenon in vitro. Here we describe a cell-free transcription elongation assay that faithfully recapitulatespoly(A) signaling in a crude nuclear extract. The assay requires the use of citrate, an inhibitor of RNApolymerase II carboxyl-terminal domain phosphorylation. Using this assay we show the following. (i) Wild-typebut not mutant poly(A) signals instruct the polymerase to stop transcription on downstream DNA in a mannerthat parallels true transcription termination in vivo. (ii) Transcription stops without the need of downstreamelements in the DNA. (iii) cis-antisense inhibition blocks signal transduction, indicating that the signal to stoptranscription is generated following extrusion of the poly(A) site from the polymerase. (iv) Signaling can beuncoupled from processing, demonstrating that signaling does not require cleavage at the poly(A) site.

It has become clear in recent years that RNA polymerase II(RNAPII) not only transcribes the mRNA but shepherds itthrough the stages of processing as well (42, 67). An earlyindicator of this coupling between transcription and processingwas the finding that the poly(A) signal for cleavage and poly-adenylation of pre-mRNA directs not only 3�-end processingof the transcript but also termination of transcription by thepolymerase (20, 32, 50, 61, 77, 79). A major challenge has beento explain how the poly(A) signal communicates with the poly-merase.

The core poly(A) signal in vertebrates consists of two rec-ognition elements flanking a cleavage-polyadenylation site (76,82). Typically, an almost invariant AAUAAA hexamer lies 20to 50 nucleotides (nt) upstream of a more variable element richin U or GU residues. Cleavage of the nascent transcript occursbetween these two elements and is coupled to the addition ofup to 250 adenosines to the 5� cleavage product. The cleavageis mediated in vitro by a large, multicomponent protein com-plex that can be separated into five distinguishable factors.Two of these factors are the cleavage and polyadenylationspecificity factor (CPSF), which binds the AAUAAA motif,and the cleavage stimulation factor (CstF), which binds thedownstream U-rich element. In vitro studies suggest that CPSF(26) and probably CstF as well (55, 71) are recruited to thepolymerase at the promoter. Presumably they then ride withthe polymerase during transcription, scanning the extrudingtranscript so as to snare the poly(A) site when it emerges. Thesituation in yeast may be similar (8, 29). Strictly speaking, theterm “poly(A) site” refers only to the point at which cleavage

occurs and the poly(A) tail is appended, but we use the termhere to refer to the poly(A) signal as a whole when this helpsto distinguish between the poly(A) signal as an entity andpoly(A) signal transduction [or “poly(A) signaling”] as a pro-cess.

Models that attempt to explain transduction of the signalfrom the poly(A) site to the polymerase can be divided intotwo categories (50): (i) cleavage-dependent models, in whichthe poly(A) site is recognized by virtue of the processing thatit carries out, and (ii) cleavage-independent models, in whichthe poly(A) site is recognized directly by a component of thetranscription elongation complex. Cleavage-independent mod-els were initially favored (50) and recently gained renewedsupport from the finding that CPSF and CstF may be compo-nents of the transcription elongation complex (55). As such,these factors would presumably be positioned to recognize thepoly(A) site as soon as it emerges so as to transduce a termi-nation signal to the polymerase. This is the mechanism used bythe vaccinia virus RNA polymerase (27, 40), which closelyresembles RNAPII in many aspects of structure and mecha-nism (see reference 28). Moreover, elongating polymerases notonly carry factors that can recognize signals in the extrudingRNA (see also reference 23), but they also have considerableintrinsic potential to recognize sequence elements at both theDNA and RNA levels prior to extrusion (4, 58). Thus, cleav-age-independent models readily offer plausible scenarios forpoly(A) signaling based on recognition of the signal eitherprior to, during, or following extrusion.

However, the preferred model for poly(A) signaling has formany years been a cleavage-dependent model in which thesignal is generated by the new, uncapped RNA 5� end resultingfrom poly(A) site cleavage (20, 68, 81). According to thismodel the new 5� end recruits a signal-transducing 5� exonu-clease which chases the polymerase by processively degrading

* Corresponding author. Mailing address: Department of Chemistryand Biochemistry, University of California at Los Angeles, Los Ange-les, CA 90095-1569. Phone: (310) 825-3767. Fax: (310) 206-4038. E-mail: [email protected].

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the RNA, thereby to deliver the signal. Consistent with thishypothesis, Edwalds-Gilbert et al. (32) and Birse et al. (14)reported a close correlation between processing efficienciesand termination efficiencies for a variety of mutated poly(A)sites and processing factors, suggesting that some aspect of theprocess leading to poly(A) site cleavage is involved in gener-ating the signal to terminate.

A different point of view has been expressed by Batt et al.(10) and Osheim et al. (61), who favored a cleavage-indepen-dent mechanism for transcription termination. However, nei-ther of these groups actually assayed termination itself. Thedata in the work of Batt et al. (10) reflected variations inprocessing efficiency rather than termination, while the poly-merase stacks in the electron micrographs of Osheim et al. (61)may well have resulted from polymerase pausing or arrest.Osheim et al. (61) pointed out that the stacked-up polymerasesin their micrographs carried mostly uncleaved, full-length tran-scripts although the stacks extended across the poly(A) site.This is actually consistent with current cleavage-dependentmodels in which cleavage triggers termination as part of asingle, concerted reaction (11, 30, 67, 80). This underscores thedifficulty until now of distinguishing experimentally betweencleavage-dependent and cleavage-independent models and haseven led to the suggestion that there is no mechanistic commondenominator to poly(A) signaling, termination being cleavagedependent, cleavage independent, or both cleavage and 5�-exonuclease dependent according to circumstance (81).

Transcriptional pause elements in the DNA downstream ofthe poly(A) signal have long played a prominent role in modelsfor poly(A)-dependent termination (2, 13, 41, 68, 81). Pausingof the polymerase is thought to be necessary to allow thepoly(A) site time to act and, thereby, to signal the polymerase.Consistent with this hypothesis pausing is indeed frequentlyobserved to occur downstream of poly(A) sites (7, 13, 16, 33,41, 53, 74, 78). Also consistent with this hypothesis, somepoly(A) sites are known to be accompanied by downstreamelements that enhance cleavage and polyadenylation and/ortermination or arrest (2, 6, 13, 21, 31, 33, 72). Although theseelements are usually referred to as pause elements because ofa presumption about their mechanism of action, it has not beenconfirmed that they actually cause the pausing seen down-stream of poly(A) sites in vivo. Moreover, pausing per se doesnot enhance cleavage and polyadenylation in vitro (80). In-deed, unlike confirmed pause elements (24, 45, 62, 65, 70),these downstream elements are not associated with pausing ina consistent way—pausing being found variously upstream,downstream, or coincident with the elements (2, 13, 33, 41).This has been attributed to DNA sequence artifacts related tothe nascent transcript analysis used to detect pausing and ter-mination (2). However, the widespread occurrence of broadzones of apparent pausing downstream of poly(A) sites sug-gests that the pausing itself is genuine and that it is the mech-anistic relationship of this pausing to the downstream ele-ments that is obscure. Thus, it is not known whether elementsin downstream DNA enhance processing and terminationthrough pausing or by some other mechanism such as by adirect effect on the poly(A) signal itself (19, 52). Nor is itknown whether the pausing commonly seen downstream ofpoly(A) sites is due to additional elements in the downstream

DNA or to an effect of the poly(A) signal itself on events thatoccur downstream.

Given the uncertainties concerning the number and natureof elements in a typical poly(A)-dependent terminator to-gether with the uncertainties concerning the basis of poly(A)dependence itself, we have adopted a parsimonious approachto the study of poly(A)-dependent termination. First, we haveconcentrated exclusively on the core of the poly(A) signal inorder to determine its unique contribution to termination. Todo this we have capitalized on our previous finding that thecore of the simian virus 40 (SV40) early poly(A) signal iscapable of inducing efficient termination in vivo without theassistance of any downstream elements in the DNA (79). Thus,we eliminate ambiguities related to the role of such elements intermination.

Second, we have limited our attention to the signaling stepof poly(A)-dependent termination—i.e., how the poly(A) sitemakes its presence known to the polymerase. The immediateconsequence of signaling need not be transcript release as isgenerally assumed. It could be pausing, consistent with theapparent collection of paused transcription complexes seen inthe striking electron micrographs of Osheim et al. (61) and assuggested by the broad pausing profiles commonly seen down-stream of poly(A) sites following nascent transcript run-onanalysis (7, 13, 16, 33, 41, 53, 74, 78). We have thereforedesigned an assay that measures the collective effects of thepoly(A) signal on the efficiency of transcription elongation.Thus, we detect the first effect of the poly(A) site on transcrip-tion [i.e., poly(A) signaling], whether that effect is completetermination, halting of the polymerase, or merely slowingdown transcription.

Finally, wishing to address questions of mechanism in adirect way, we have devised an in vitro system for studyingpoly(A) signaling. There have been prior reports suggestingsuccessful coupling in vitro of polyadenylation to termination.However, one such report (57) actually described measure-ments not of termination but of poly(A) site cleavage, whileanother (81) failed to show that the termination under studywas dependent on the presence of a poly(A) signal. In contrast,we now describe an in vitro system that faithfully reproducessignaling from the poly(A) site to the polymerase, and wevalidate this in vitro assay by reference to an in vivo assay thatis free of the potential artifacts associated with conventionalhybridization-based nascent transcript analysis (79). We showbelow that signaling in vitro does not require pause elements inthe DNA and does not require cleavage of the transcript butdoes require extrusion of the poly(A) site from the polymerase.This is the first successful demonstration of poly(A) signalingin vitro.

MATERIALS AND METHODS

Plasmids. In our nomenclature, subscripts (as in APw and APm) refer toplasmids with wild-type and mutant poly(A) signal hexamers, respectively. Oftenthe subscripts are omitted when the context is clear or when mutant and wild-type versions of the plasmid are being referred to collectively or generically.Brackets (as in �cat�) refer to G-less cassettes of 120 and 261 bp flanking theindicated sequence unless otherwise specified, as in �117cat�. Plasmids not de-scribed elsewhere were constructed as follows. For pAPw�117cat�, a BamHI sitewas introduced into the 377-bp precassette of pAP�cat� (79) by site-directedmutagenesis, and the resulting 323-bp BamHI fragment was removed, leaving a117-bp precassette. pAPm�117cat� was constructed as described above except

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that the poly(A) signal was first inactivated by mutating the hexamer (AATAAA3AgTAct [lowercase letters indicate mutations]) using site-directed mutagen-esis. For pAP�cat�, SmaI and HindIII sites were added at the 3� end of theupstream cassette (simultaneously lengthening it to 120 bp) in the pAP�117cat�plasmids by use of site-directed mutagenesis (gcttggcgagatt3cccgggcaagctt). ForpAP�377�, the pAP�cat� plasmids were cut with HindIII and EcoRV and theBamHI-RsaI fragment containing the 377-bp G-less cassette from pAP�cat� wasinserted. The construction of pAP�C9� was like that for pAP�377�, but the insert,C9, was a 1,012-bp piece of DNA consisting primarily of nine copies of an arbi-trarily selected 102-bp sequence from the middle of the C2 region of �-globin3�-flanking DNA (79), containing no known functional elements. Forp377A174P�C9�, the 377-bp G-less cassette (above) and a 174-bp cassette wereinserted into pAP�C9� at the StuI and HpaI sites, respectively. The 174-bpcassette was obtained by cutting pAP�cat� with BglII and HinfI after site-directedmutagenesis (atattt3aGatCt) of the 261-bp cassette. For pAP�C9377�, the1,012-bp C9 fragment was inserted into SmaI-cut pAP�377�.

Poly(A) signaling assay. HeLa nuclear extract was prepared exactly as de-scribed by Flaherty et al. (34) except that the final centrifugation was at 13,000 �gav for 30 min. The extract was dialyzed against buffer D until the conductancesof the extract and the buffer were equal (5 to 7 h). The extract was then aliquotedand stored in liquid nitrogen. Different extracts vary somewhat in their proper-ties.

A typical signaling assay began with 6 to 12.5 �l of nuclear extract, which wasbrought to a total volume of 12.5 �l with buffer D. This was then mixed withsodium citrate, dithiothreitol, creatine phosphate, MgCl2, anti-RNase (Ambion),DNA, and water up to a volume of 22 �l. The mixture was preincubated at 30°Cfor 30 min, and then 3 �l of nucleoside triphosphate mix containing 20 �Ci of[�-32P]CTP (800 Ci/mmol) was added and transcription was allowed to continuefor 15 min. Final concentrations in a typical transcription mixture were as fol-lows: 10 mM HEPES (pH 7.9); 10% glycerol; 50 mM KCl; 0.1 mM EDTA; 2 mMdithiothreitol; 8 mM sodium citrate (pH 6.7); 20 mM creatine phosphate; 5 mMMgCl2; 15 to 30 U of anti-RNase; 0.6 �g of DNA; a 200 �M concentration (each)of ATP, GTP, and UTP; and 5 �M unlabeled CTP. Any variations from thisstandard procedure are noted in the figure legends. Because of some variationsbetween experiments, including the use of several different extract preparations,comparisons of absolute signaling efficiency should be made only within, notbetween, figures.

For most of the experiments reported in this work, transcription was stoppedby addition of 1 �l each of �-amanitin (1 mg/ml; Sigma) and DNase I (2 U/�l;Ambion). After 10 min at room temperature, 1 �l each of 50 mM EDTA andRNase T1 (200 to 250 U/�l; Ambion) was added for 15 min at 30°C. Then 1 �lof proteinase K (20 mg/ml) was added and, after 10 min at room temperature,the reaction was extracted with 350 �l of TRIzol (Gibco BRL) and 70 �l ofchloroform. However, a simpler procedure is often adequate, in which transcrip-tion is stopped using 2 �l of 250 mM EDTA containing 200 U of RNase T1,followed, after 15 min, by TRIzol extraction. The RNA was precipitated with 1.5to 2 �l of Saccharomyces cerevisiae tRNA (10 mg/ml; Sigma) and 350 �l ofisopropanol (10 min, room temperature), spun in a microcentrifuge (10 min,4°C), washed with 150 �l of 75% ethanol, resuspended in 15 �l of 7 M urea-loading dye, heated at 90°C for 5 min, chilled immediately on ice for 1 min, andloaded onto an 8% polyacrylamide gel to separate the G-less transcriptionproducts. For analyzing transcripts containing antisense sequences, one or twoadditional digestions with T1 under denaturing conditions were carried out, asfollows. Instead of resuspending in urea after the first precipitation, the RNAwas resuspended in 32 �l of 50% formamide–0.5 mM Tris–0.5 mM EDTA (pH8) and then heated to 90°C for 5 min. After cooling to 37°C, 7 �l of RNase T1

(1,000 U/�l) was added and the samples were placed in an oven at 63°C for 1 h.Extraction with TRIzol and subsequent steps were then carried out as describedearlier.

Following electrophoresis, results were recorded and analyzed using a Phos-phorImager with ImageQuant software (Molecular Dynamics). Unless otherwiseindicated, results are reported as the average of two separate experiments carriedout on different days. Error bars indicate the range of values obtained in the twoindividual experiments. For most individual experiments, reactions were carriedout in duplicate and the averages of the duplicates were taken as the outcome ofthe experiment.

Nascent transcript G-less cassette analysis of poly(A)-dependent terminationin vivo (79). COS cells were grown in 35-mm-diameter wells and transfected withplasmid DNA using Fugene 6 (Roche). After 48 h cells were rinsed twice withcold phosphate-buffered saline and lysed with a solution containing 0.5%IGEPAL (Sigma), 10 mM Tris (pH 7.4), 3 mM MgCl2, and 10 mM NaCl. Nucleiwere pelleted and then resuspended in 15 �l of 50 mM Tris (pH 8.3)–0.1 mMEDTA–40% glycerol–5 mM MgCl2. Run-on transcription was carried out for 45

min at 30°C following addition of 16 �l of a solution containing 10 mM Tris (pH8), 5 mM MgCl2, 600 mM (NH4)2SO4, 1 mM ATP, 1 mM UTP, 0.2 mM3�-OMeGTP, 6 mM dithiothreitol, 20 U of anti-RNase, and 1 �l of 180 �M CTPcontaining 30 �Ci of [�-32P]CTP. After a 10-min cold chase with 1 �l of 40 mMCTP, 1 �l (15 U) of T1 RNase and 1 �l of 50 mM EDTA were added for anadditional 30 min at 30°C. Following digestion with 2 �l of DNase I (20 U) for15 min at 30°C, 1.8 �l of 10% sodium dodecyl sulfate was added. The sample wasthen extracted with 500 �l of TRIzol and 140 �l of chloroform, and 20 �g ofcarrier tRNA and 500 �l of isopropanol were added to the aqueous phase toprecipitate the RNA. The RNA was resuspended in 32 �l of 10 mM Tris–1 mMEDTA (pH 7), heated for 2 min at 90°C, and cooled on ice for 1 min, and then1 �l (250 U) of T1 was added for 30 min at 30°C. TRIzol (350 �l) extraction wasthen carried out in a manner similar to that described above.

RNase protection assay. RNA from the equivalent of four standard signalingassays (with cold CTP replacing [�-32P]-CTP) or transfected RNA was analyzedas described (18) with hybridization carried out at 63.5°C. The probe used was aT7 RNA polymerase transcript of BglI-digested pAP�cat� into which the T7promoter (HincII-PvuII fragment from pBluescript II SK [Stratagene]) had beeninserted at the HindIII site.

RESULTS

Poly(A) signaling in vitro mimics poly(A)-dependent termi-nation in vivo. Poly(A) signaling in our system is detected as adecrease in the elongation efficiency of RNAPII after crossinga poly(A) site. To measure signaling we modified a G-lesscassette assay previously used to monitor the efficiency of tran-scriptional elongation in vitro (48, 51). Figure 1 illustrates theuse of this assay to study signaling during transcription innuclear extracts and compares the results obtained for signal-ing in vitro with parallel results obtained for termination invivo.

Figure 1A shows a diagram of pAP�C9377�, the DNA tem-plate used for these experiments. It contains the core SV40early poly(A) signal followed immediately by a short, 120-ntG-less cassette. Two additional cassettes, distinguishable insize, lie farther downstream, separated from the upstream cas-sette by a spacer sequence (called C9). The efficiency of elon-gation downstream of the poly(A) site is indicated by theextent to which the two downstream cassettes (postcassettes)are transcribed relative to the upstream cassette (precassette).Poly(A) signaling is evident when this elongation efficiency isless for templates with wild-type poly(A) sites than for tem-plates with mutant poly(A) sites (hereafter called mutant orwild-type templates).

Figure 1B, lane 1, shows the G-less RNAs that surviveRNase T1 digestion of the transcripts produced in vitro fromthe wild-type version of the pAP�C9377� template. Lane 2shows the results, obtained in parallel, for the mutant versionof the template which differs only in that the AATAAA hex-amer of the poly(A) signal has been mutated to AgTAct.Clearly the downstream cassettes are transcribed less effi-ciently from the wild-type template than from the mutant tem-plate. The results are presented quantitatively to the right ofthe gel images. The molar ratio of 261-nt postcassette tran-scripts to 120-nt precassette transcripts is 0.51 for the mutantbut only 0.19 for the wild-type template. Thus, transcriptionalelongation on the wild-type template between the 120- and the261-nt cassettes is only 37% as efficient as on the mutanttemplate. In Fig. 1B and C, and throughout this report, werefer to this as the percent wild-type readthrough.

We note that the postcassette/precassette molar ratio is lessthan one even for the mutant template. This may reflect, in

VOL. 21, 2001 POLY(A) SIGNAL TRANSDUCTION TO RNA POLYMERASE IN VITRO 7497

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part, frequent pause and/or arrest of RNAPII induced by thelow concentrations of CTP in our transcription mixtures (66)combined with protein binding to the DNA in our extracts andan insufficient concentration of TFIIS to efficiently overridethese nonspecific impediments to transcription (47). The lowpostcassette/precassette ratio also reflects our use of relativelyshort transcription times for these experiments. We have cho-sen to concentrate our attention on the forward wave of poly-merases that elongate most efficiently down the template,which means that many stragglers and late-initiating poly-

merases will have transcribed the precassette but not yet ar-rived at the postcassette by the time that we stop the reaction.However, these effects of transcription time and CTP concen-tration do not compromise our conclusions, because they applyto both wild-type and mutant templates equally and are thusnormalized away when we take the wild-type/mutant ratios.Control experiments show that none of our conclusions de-pends on the particular transcription time or concentration ofCTP chosen.

The ability of the wild-type poly(A) signal to decrease re-covery of the downstream cassettes relative to the upstreamcassette reflects a decrease in downstream transcription on thewild-type template. The reduced recovery is not a consequenceof preferential degradation of the downstream cassettes fol-lowing 3� end processing of the wild-type transcript. This con-clusion follows from the design of the template: all three cas-settes in pAP�C9377� lie downstream of the poly(A) signal(Fig. 1A). Thus, although processing would indeed destabilizedownstream RNA, this would include all of the cassettes.Moreover, the upstream cassette would, if anything, be more,not less vulnerable to degradation since degradation is proba-bly mediated by a 5�-specific exonuclease (60, 81). Therefore,the observed effect must be transcriptional, not posttranscrip-tional, in nature (see also below).

Next we asked whether the poly(A) signaling observed invitro (Fig. 1B) is related to poly(A)-dependent termination asmeasured in vivo (79). To measure termination in vivo wetransfected mutant and wild-type pAP�C9377� into COS cells.We then harvested nuclei after 2 days and carried out nascent-transcript analysis to determine the steady-state distribution ofpolymerases over the three G-less cassettes in vivo (79). Itshould be emphasized that this nascent-transcript G-less-cas-sette analysis reflects actual termination in vivo (not pausing)and is free of many of the potential artifacts associated withconventional run-on transcription-hybridization analyses (seereference 79 for a discussion). Figure 1C is a plot of theefficiency with which polymerases reach the downstream cas-settes on the wild-type compared to the mutant templates bothin vivo and in vitro. Clearly the signaling that occurs in vitroclosely parallels the termination that occurs in vivo. We con-clude that signaling either yields termination directly, or ittriggers an event such as pausing that leads to termination withhigh efficiency.

Poly(A) signaling commences at the poly(A) site. The resultsof Fig. 1 show that transcription is impaired downstream of awild-type poly(A) site. The simplest interpretation is that thepolymerases become transcriptionally impaired as a conse-quence of crossing the poly(A) site. However, it is formallypossible that the wild-type template differs from the mutanttemplate in some global property (e.g., supercoiling) that im-pairs transcription throughout the plasmid. To distinguish be-tween these possibilities, we assayed transcription both up-stream and downstream of the poly(A) site on mutant andwild-type versions of the p377A174P�C9� template. This plas-mid contains four cassettes, two upstream and two downstreamof the poly(A) site (Fig. 2).

First, we confirmed that the four cassettes in this plasmid aretranscribed successively, as predicted for polymerases thatinitiate at the SV40 early promoter, and then traverse eachcassette in turn. To avoid any effect on transcription from

FIG. 1. Poly(A) signaling in vitro. (A) The pAP�C9377� constructdrawn to scale. The arrows indicate the start point of transcription forthe SV40 early-early promoter (9) and the position of poly(A) sitecleavage (20). (B) G-less cassette transcripts from a signaling assaycarried out with mutant and wild-type versions of pAP�C9377�. Theline graphs showing signal intensities for the two gel lanes were super-imposed, but offset slightly for clarity, and then aligned with the gelimages. (C) Quantitative comparison of poly(A) signaling in vitro withpoly(A)-dependent termination in vivo on the pAP�C9377� template.The efficiency of elongation (% wild-type readthrough) downstream ofthe wild-type poly(A) site is expressed (after normalizing all signals oneach template to that for the 120-nt cassette) as the ratio of theintensity for each cassette on the wild-type template to that for thesame cassette on the mutant template. (The images in panel A werealso normalized with respect to the 120-nt cassette for purposes ofpresentation.)


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an active poly(A) site we used the mutant version ofp377A174P�C9� for these preliminary experiments. Figure 2A(top panel) shows that the cassette RNAs do indeed appearsequentially during transcription and then build in concentra-tion. In the bottom panel of Fig. 2A the cassette RNA con-centrations are expressed relative to that of the first cassette inthe series to emphasize their sequential appearance. Figure 2Bshows that the cassettes upstream of the poly(A) site are tran-scribed similarly from the mutant and wild-type templates butthat, downstream of the poly(A) site, transcription on the wild-type template rapidly falls off. Thus, transcription on the wild-type template diminishes relative to the mutant only after thepolymerases cross the poly(A) site.

Different poly(A) sites signal similarly. Figure 3 summarizesresults showing that in addition to P (the core SV40 early site),both S (SPA of reference 49) and L (the complete SV40 latesite) carry out signaling in our assay. Thus, pAPw�C9377� ex-hibits impaired elongation compared to its mutant (Fig. 3,constructs 1 and 2) as we have already shown (Fig. 1). How-ever, when the mutant poly(A) site of pAPm�C9377� was re-placed, following HpaI-BamHI digestion, by either wild-type S(construct 3) or wild-type L (construct 4), signaling was re-stored. Thus, the ability of the system described here to sup-port signaling in vitro is quite general.

Characteristics of the signaling reaction. The most strikingcharacteristic of the in vitro poly(A) signaling reaction is thecitrate requirement. This is illustrated in Fig. 4A where theefficiency of signaling (i.e., 100 % readthrough) is plotted asa function of citrate concentration. Signaling is not observed inthe absence of citrate, but as the citrate concentration is in-

creased (in the presence of 7 mM Mg2� for this extract) sig-naling rises and goes through a maximum at a citrate concen-tration of about 8 mM (Fig. 4A). Citrate curtails RNAPIIcarboxyl-terminal domain (CTD) phosphorylation and reducesthe elongation efficiency of transcription in vitro (64). We weredrawn to the use of citrate in the signaling reaction because ofthe role of citrate in allowing the transactivation response(TAR) element in nascent human immunodeficiency virus(HIV) transcripts to mediate an increase in HIV transcriptionelongation efficiency (64). In the absence of citrate, apparently,the intrinsic elongation efficiency of HIV transcription is toohigh to be augmented by TAR. Similarly, after a long period offruitless experimentation, we reasoned that perhaps the intran-sigence of our system reflected an intrinsic transcription elon-gation efficiency that was too high in vitro to be abrogated bythe poly(A) signal.

The results in Fig. 4A show how the relationship betweensignaling and elongation efficiency varies as a function of ci-

FIG. 2. Transcription is impaired only downstream of the poly(A)site. (A) Cassettes are transcribed sequentially from the SV40 earlypromoter. Transcription of the mutant version of p377A174P�C9� wascarried out as described in Materials and Methods except that 7 mMMg2� and 7 mM citrate were used, and transcription times were variedfrom 1 to 32 min. The top panel shows molar amounts of each cassetteproduced plotted on an arbitrary scale. The bottom panel shows molarratios of each cassette produced relative to the 377-nt cassette. (B)Elongation efficiency drops after crossing the poly(A) site. The percentreadthrough for each cassette on the wild-type template relative to thatof the mutant is plotted, as for Fig. 1C, above a scale map of thetemplate.

FIG. 3. Poly(A) signaling by different poly(A) sites. ConstructpASw�C9377� was obtained by replacing the mutant poly(A) site ofpAPm�C9377� with an HpaI-BamHI fragment differing only at the endsfrom the SPA sequence used previously (18): 5�-aacaataa. . . tgtgtctagaactagtg-3�. Similarly, for pALw�C9377� the mutant poly(A) site wasreplaced with a SmaI-BamHI-trimmed PCR copy of the SV40 late sitediffering only at the ends from that used previously (18): 5�-ggggatctggac. . . tgggag-3�. The histogram shows the percent readthrough for the261-nt cassette of each template relative to that of pAPm�C9377�. Forthe extract preparation used in these assays the citrate and Mg2�

concentrations were both 7 mM.


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trate concentration. The hypothesized inverse correlation be-tween these two parameters is evident between 4 and 8 mMcitrate as the signaling curve rises to its maximum. At highercitrate concentrations (10 and 12 mM) signaling declines. Thisdecline may reflect, in part, the ability of citrate to chelateMg2�, since the detection of signaling in our system is im-paired at low [Mg2�] (Fig. 4B and C).

Figure 4A also confirms that the effect of citrate on tran-scription is principally at the level of elongation rather thaninitiation, since the elongation efficiency curve closely matchesthe curve for total transcription in the range of 4 to 12 mMcitrate. It is not surprising that elongation efficiency is an im-portant determinant of cassette yield since, for templates suchas pAP�C9377�, even the first cassette lies nearly 800 bp down-stream of the start point of transcription. The curve for totaltranscription drops below that for elongation efficiency at 0mM citrate, probably because initiation is impaired at thehigher free Mg2� concentration that exists in the absence ofchelation by citrate. This [Mg2�] is considerably higher thanthe optimum for the SV40 early promoter (2 mM) in ourextracts (data not shown). A similar effect is apparent in Fig.4B and C, where the curve for total transcription decreases athigh Mg� concentrations despite continuing increases in elon-gation efficiency.

In addition to citrate, the signaling reaction at its presentstate of development requires that the nuclear extract be verycrude. As yet we have not subjected this parameter to carefulscrutiny, but consistently we have obtained the best signalingwith extracts subjected only to a relatively low-speed spin fol-lowing extraction of the nuclei (see Materials and Methods).The state of the DNA (linear or supercoiled), its method ofpurification (spin column, gel, or CsCl), and the presence orabsence of DNA or RNA carrier are unimportant to signaling.Cassette signals are completely �-amanitin sensitive at 0.2�g/ml (data not shown) and therefore arise entirely fromRNAPII transcription. In summary, poly(A) signaling in our invitro system is robust and reproducible and therefore suitablefor use in mechanistic studies.

Poly(A) signaling resembles a stochastic process and re-quires no special element in the downstream DNA. The data inFig. 1C suggest that the ability to elongate is lost increasinglywith distance after crossing the poly(A) site. This trend isillustrated further in Fig. 5, which shows examples of experi-ments that are representative of our experience with the sys-tem. In Fig. 5 the histogram peaks have been aligned with thedistal ends of their corresponding cassettes to reflect the dis-tances that must be traveled by the individual polymerases toproduce the various cassette transcripts. The data show thattranscription gradually decreases across more than 1.8 kb ofDNA downstream of the core SV40 early poly(A) site. Wehave shown previously that commitment of the SV40 earlypoly(A) site to cleavage and polyadenylation in vivo is a sto-chastic process (18). In this respect, signaling by this poly(A)site to stop transcription appears to be similar.

FIG. 4. Citrate and Mg2� titrations of the signaling reaction. Totaltranscription refers to the yield, in arbitrary PhosphorImager units, ofthe 120-nt cassette from the mutant template. It is called total tran-scription because both initiation and elongation efficiencies contributeto the yield. Elongation efficiency is defined here as the efficiency of120- to 261-nt cassette readthrough, in this case when signaling isabsent (i.e., the molar ratio of 261- to 120-nt cassette transcription onthe mutant template). Signaling is defined as 100% % wild-typereadthrough (i.e., 100 minus the wild type-versus-mutant elongationefficiency ratio, expressed as a percentage). Points without error barscorrespond to values taken from a single experiment only. Severaldifferent extract preparations were used for the experiments reportedin this paper. Therefore, the citrate and Mg2� optima exhibited in thisfigure differ slightly from the concentrations used for other experi-ments reflecting the use of a different extract preparation here. Also

note in panels B and C that equivalent signaling efficiencies can beobtained using different combinations of citrate and Mg2� concentra-tions, further accounting for the use of different concentrations ofthese constituents in different experiments.


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Significantly, the poly(A)-dependent decrease of transcrip-tion for the templates in Fig. 5 appears to be relatively insen-sitive to the nature of the downstream DNA. Thus, for thepAP�377� template, substantial poly(A)-dependent loss oftranscription occurs across a region that contains no eukaryoticDNA. In this template the DNA contains, for more than 1 kbdownstream of the poly(A) signal, only a mixture of artificialsequences (G-less cassettes and multiple cloning sites) andprokaryotic DNA (chloramphenicol acetyltransferase andpBR322). In contrast, the DNA between the cassettes inpAP�C9� is mostly eukaryotic in origin, having been obtainedfrom the transcription termination region of the chicken �H

globin gene. Yet both templates fit the same trend for decreaseof transcription with distance following the poly(A) site. Thislack of sensitivity to the nature of the underlying DNA isfurther underscored by the results for pAP�cat� of Fig. 6. Herethe poly(A)-dependent loss of transcription takes place acrossyet a different downstream DNA sequence, but the effect stillfits the trend shown in Fig. 5. Since DNA from eukaryotic

termination regions, prokaryotic coding regions, and artificialconstructs all support signaling similarly, we conclude thatsignaling in vitro does not require any special element in thedownstream DNA.

Poly(A) signaling does not occur until after poly(A) siteextrusion. It is generally assumed that poly(A) signaling occursafter the poly(A) site has been extruded from the polymerase.This supposition has never been tested experimentally and isbased on the presumption that the poly(A) signal is too com-plex to be recognized directly by the transcription apparatus(32). However, Escherichia coli RNAP is able to monitor DNAsequence throughout its 35-bp footprint to receive regulatoryinput for both pausing and termination (4, 58). The DNAcontacts for RNAPII are presumably even more extensive (25).Additional opportunities for intrinsic regulatory input comefrom interactions of the RNAP with the DNA-RNA hybridand with the RNA itself in the exit tunnel (58). Moreover, theefficiency of poly(A)-dependent termination is not, as previ-ously thought (32), directly related to processing efficiency (1,79), indicating the existence of additional sources and/orsources of regulatory input different from simply the efficiencyof processing. Although processing factors are known to affectsignaling (8), their role may be limited to maintaining thetranscription complex in a responsive conformation. Thus, cur-rent data are not inconsistent with a model in which poly(A)signaling reflects an intrinsic ability of the transcription appa-ratus to recognize the poly(A) site. We therefore decided totest this possibility directly.

If the transcriptional apparatus is intrinsically capable ofrecognizing the poly(A) site, then signaling may occur beforethe poly(A) site is extruded from the polymerase. To deter-mine whether poly(A) signaling occurs before or after extru-sion of the poly(A) site, we used the cis-antisense approach.This method involves cloning an inverted copy of a poly(A)signal downstream of the properly oriented parent and hasbeen used to study the timing of cleavage and polyadenylationcomplex assembly in vivo (18). Transcription of the invertedpoly(A) signal generates an antisense transcript to the authen-tic poly(A) signal upstream. When this antisense transcriptemerges from the polymerase, it forms a duplex with the pre-viously extruded poly(A) signal and prevents cleavage andpolyadenylation (18). If signaling to the polymerase, like as-sembly of the cleavage and polyadenylation apparatus, occursfollowing extrusion of the poly(A) site, then signaling-like pro-cessing may be blocked by the antisense transcript. However, ifsignaling occurs earlier—during transcription and extrusion—then the antisense should be unable to block an event that hasalready occurred.

To determine whether an antisense transcript to the poly(A)site can block signaling to the polymerase, we prepared theconstructs diagrammed in Fig. 6A. As before, each constructshown refers to a mutant and wild-type pair. Construct 1,pAP�cat�, was the parent construct for the plasmids of thisexperiment. The separation between the cassettes in pAP�cat�is slightly greater than in pAP�377� of Fig. 5, and pAP�cat�exhibits a slightly greater poly(A)-dependent decrease in tran-scription; readthrough for the wild type is �60% of that for themutant (Fig. 6A, construct 1). Construct 2 contains, just down-stream of the 120-nt cassette, a 218-nt antisense module (�)which is an inverted repeat of the poly(A) signal and its up-

FIG. 5. Transcription diminishes gradually with distance followingthe poly(A) site. Poly(A) signaling on the pAP�C9377� template (Fig.1B) is compared with that of two other templates having cassettesseparated by shorter distances. Only single examples of reactions withpAP�377� and pAP�C9� are presented here. These reactions were car-ried out under conditions similar to those described for Fig. 1B andcould therefore be directly compared. However, the conclusions forthese constructs are consistent with numerous other experiments car-ried out under a variety of slightly different conditions (that preventtheir being plotted on the same graph).


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stream flanking sequences. The antisense transcript is targetednot only to the poly(A) signal (whose cleavage site is 19 ntupstream of the 120-nt G-less cassette [Fig. 6B]) but also tosome upstream flanking sequence to ensure adequate duplexstability when the stem-loop (Fig. 6C) forms in the RNA (18).

Figure 6A shows that construct 2 exhibits no signaling: wild-type transcription is indistinguishable (99% readthrough ratio)from that of the mutant. Thus, the presence of the antisensetranscript completely blocks signaling by the poly(A) site. This

is despite the fact that the separation between the cassettes inconstruct 2 is significantly greater than in construct 1 so that,other things being equal, signaling for construct 2 would havebeen expected to be greater than for construct 1 (Fig. 5).

Notice, for construct 2, that the nascent RNA must be ex-tended more than 126 nt beyond full extrusion of the poly(A)signal from the polymerase before the antisense transcripteven begins to appear (Fig. 6A, construct 2, and B and C).Moreover, since the antisense must be extruded a further 10 to

FIG. 6. Signaling is not an early event following poly(A) site transcription. (A) Signaling can be inhibited by an antisense transcript placed 126nt downstream of the poly(A) site. The antisense segment (open arrow) is the HinfI-BamHI fragment of the sequence in panel B insertedbackwards into either HindIII- or EcoRV-cut pAP�cat�. The region targeted by the antisense sequence is denoted by the grey arrow. Thesense-antisense separations for constructs 2 and 3 are 126 and 686 nt, respectively. (B) Sequence for the region denoted by the square bracket overconstruct 2 in panel A. The sequence for the wild-type version, pAPw��cat�, is shown. The complete sense region (underlined) and part of theantisense region (also underlined) are presented. The poly(A) signal and its downstream complement are shown in boldface type. The G-lesscassette is shown in lowercase type. (C) Diagram, drawn linearly to scale, of the stem-loop structure predicted to form in the sense-antisense regionof transcripts from the mutant version, pAPm��cat�, of construct 2. The tick marks above and below the duplex indicate the positions of G residues.Note that since it is the mutant version of the stem-loop that is shown, there is a G in the hexamer region of the sense strand reflecting the presenceof the hexamer mutation (see Materials and Methods). Accompanying this mutation is a small interruption of base pairing in the stem at theposition of the hexamer since the wild-type sequence was used for the antisense module in all of our constructs. The loop is comprised of 117 ntfrom the 120-nt G-less cassette plus 9 nt of non-G-less sequence at its 3� end. Three nucleotides at the 5� end of the G-less cassette invade thesense-antisense duplex. Multifold secondary structure analysis (44) predicts that the three G residues flanking the 3� end of the cassette interactwith a patch of C’s (shown in white) near its 5� end. This secondary structure model for the sense-antisense stem-loop predicts more efficient cuttingby RNase T1 in sequences flanking the base of the stem (dark arrow) than within its loop (open arrow) and slower cutting still (dotted arrow) atthe mispaired G residue in the mutated hexamer (38). (D) RNase T1 digestion confirms the predicted secondary structure of the pAPm��cat�stem-loop. Transcription of pAPm�cat� and pAPm��cat� was carried out for either 15 or 2 min as indicated. Citrate (6 mM) was used. Followingtranscription one-fourth volume of RNase T1 (1,000 U/�l) was added for 0.25 min or 1.25 min as indicated, and then the mixture was extractedwith TRIzol and the RNA was analyzed by gel electrophoresis. The interpretive drawings in the middle of the panel refer to the stem-loop structureshown in part C cut by T1 at one or more of the indicated positions. The mobilities of G-less markers and their nominal sizes are shown on theleft. The true sizes of the G-less markers are greater by one than the nominal sizes owing to the single G that remains at the 3� end following cuttingby T1.


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20 nt before sense-antisense duplex formation can even beinitiated, we conclude that the signaling normally detected byour assay does not occur until at least 140 nt after extrusion ofthe poly(A) site. In fact, the estimate of 10 to 20 nt as theminimum length for initiation of duplex formation is almostcertainly a considerable underestimate. Not only must the ini-tiating duplex be stable enough to form across a large and fairlyunstructured G-less RNA loop, but it must also be stableenough to compete with factors seeking to bind the poly(A)site. It is, therefore, likely that the bulk of the signaling de-tected in these assays actually does not occur until beyond 140nt following poly(A) site extrusion.

To determine whether the antisense module might be exert-ing some unspecified negative effect on signaling for this tem-plate, we also assayed signaling using construct 3 of Fig. 6A.This construct differs from construct 2 only in that the anti-sense module is located several hundred base pairs fartherdownstream, near the end of the interval over which signalingis measured. Since construct 3 is identical to construct 1 up tothe beginning of the antisense module, signaling for the twoconstructs should be similar if the presence of the antisensemodule in the template is benign. As shown in the histogram ofFig. 6A, signaling for construct 3 did indeed resemble that forconstruct 1, confirming that the mere presence of the antisensesequence in the template is not detrimental to the signalingassay.

Taken together the above cis-antisense inhibition resultsdemonstrate for the first time that the signal to stop transcrip-tion occurs at the level of the RNA and that signaling followsextrusion of the poly(A) site from the polymerase. Indeed,signaling occurs more than 140 nt after the poly(A) signal hasbeen completely extruded (Fig. 6A), indicating that signaltransduction occurs sometime in the interval between 140 ntpostextrusion and the time at which the transcript is cleaved.

Confirmation of sense-antisense duplex formation duringtranscription in vitro. The interpretation of the above cis-antisense results assumes sense-antisense duplex formation.Numerous control experiments in vivo support this mechanism(18). Below we confirm, by means of RNase T1 secondarystructure mapping, that sense-antisense duplex formation alsooccurs rapidly during transcription in vitro. Indeed, the G-lesscassette analysis for Fig. 6A required RNase T1 digestion un-der denaturing conditions (T1 is single-strand specific) in orderto digest the duplexed RNA produced by constructs 2 and 3(see Materials and Methods).

To assess sense-antisense duplex formation during transcrip-tion we carried out the secondary structure mapping directly inthe transcription mixtures (Fig. 6D). For this mapping we usedpAPm�cat� and pAPm��cat�, which are the mutant versions ofconstructs 1 and 2. The mutants were chosen in order to elim-inate any differences in transcription efficiency between theconstructs due to signaling. Immediately following transcrip-tion we subjected the transcription mixtures to a 1.25-minpulse of RNase T1 digestion. Figure 6C shows the structure ofthe sense-antisense stem-loop that is predicted to form in tran-scripts of pAPm��cat�. RNase T1 is expected to cleave rapidlyin the unstructured RNA to the right of the stem-loop, moreslowly in the loop itself, and slowest of all at the mismatched Gin the mutated hexamer (see Fig. 6C legend). Thus, T1 diges-tion is predicted initially to excise an intact stem-loop and then

to process it further by cutting between the cassette and theantisense sequence and at the mismatched G (Fig. 6C). Thecassette can be cleaved from the sense sequence only whenfraying at the end of the duplex, to which it is anchored,permits. On a denaturing gel these products would be expectedto run as 564-, 336-, 222-, 175-, and 161-nt species, accompa-nied by a small amount of 120-nt G-less cassette (see interpre-tive drawings in Fig. 6D). The results in Fig. 6D, lane 2, con-firm these predictions. The presence of each predicted speciesis apparent, together with a low yield of the 120-nt cassette. Incontrast, lane 1 shows that for the homologous plasmid withoutthe antisense no species other than the 261-nt and 120-ntG-less cassettes appear. When we reduced the T1 pulse to 0.25min, the 564-nt stem-loop band increased and the 120-nt G-less cassette band decreased in intensity (Fig. 6D, lane 4) asexpected for a precursor-product relationship. The vanishing120-nt band in lane 4 suggests that stem-loop formation isquantitative. The shorter T1 pulse was also accompanied byincomplete trimming of the cassette bands themselves (lane 3).Similar results have been obtained using 1/10 the concentra-tion of T1 and longer digestion times.

The band assignments in Fig. 6D have been confirmed basedon several independent criteria. First, the bands appear onlyfor constructs that produce transcripts containing antisense.Second, the bands resulting from secondary structure are se-lectively eliminated by T1 digestion under denaturing condi-tions. Third, the mobilities of the bands are consistent with thesizes predicted from Fig. 6C. Finally, the mobilities of theindividual bands can be selectively varied by targeted sequencealterations in the predicted stem-loop (data not shown). Thus,(i) the positions of the bands at 336 and 161 nt can be selec-tively varied by altering the size of the G-less cassette; (ii) thebands at 175 and 161 nt are selectively absent when the exper-iment is done with pAPw��cat�, which has no mismatched G atthe hexamer position; (iii) the position of the band at 222 ntvaries with the length of the antisense module used; and (iv)the G-less cassette band at 120 nt is no longer underrepre-sented if a G-containing insert (which can be cut by T1) isplaced between the sense strand of the predicted duplex andthe cassette.

The data above confirmed the efficient formation of thepredicted sense-antisense stem-loops by the end of a 15-mintranscription. To determine whether these structures formcontinuously during transcription we repeated the 0.25-min T1

analysis, but after only 2 min of transcription. Figure 6D, lane6 shows that the same spectrum of stem-loop digestion prod-ucts is produced within this short period of transcription as isproduced during the standard reaction (lane 4). Taken to-gether these control experiments show that sense-antisenseduplex formation occurs efficiently and continuously duringtranscription in vitro, as required by a cis-antisense inhibitionmechanism.

The signal to stop transcription is generated before tran-script cleavage. As pointed out earlier, the differential cassetterecovery that results from attenuated transcription (i.e., re-duced production of postcassette) is the opposite of that ex-pected from the degradation that accompanies 3�-end process-ing (i.e., preferential loss of precassette). Thus, by using thedifferential recovery of cassettes as a criterion in the develop-ment of the in vitro signaling assay, we simultaneously selected


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for conditions that would uncouple processing from signaling.This is apparent in Fig. 4B and C, where signaling peaks atrelatively high Mg2� concentrations and is severely depressedat the low Mg2� concentrations that favor cleavage in vitro (56,59). If signaling and processing have indeed been uncoupled inour system, that would indicate that signaling does not dependon transcript cleavage.

To determine directly whether signaling and processing areuncoupled in our system, we made a new construct for studyingsignaling, pA�G178

catC9�, as shown in Fig. 7A. This constructwas explicitly designed so that the assay could work only ifprocessing is not a prerequisite for signaling. The essence ofthe design is a new 178-bp upstream cassette containing animbedded G-less poly(A) signal (Fig. 7A). This cassette pro-vides not only the upstream cassette for signaling analysis butalso the poly(A) site itself. Since the poly(A) site is imbeddedin the G-less cassette, any instance of poly(A) site cleavage willalso result in cassette cleavage. Thus, if poly(A) signaling de-pends on prior poly(A) site cleavage, then every failure of the261-nt cassette to be transcribed (because of signaling) will bematched by a failure of the 178-nt cassette to be recovered(because of cleavage). That is, if cleavage is required forpoly(A) signaling, no decrease in 261-nt cassette recovery rel-ative to recovery of the 178-nt cassette will be apparent as aresult of signaling, and the basis for detection of signaling inour assay will be eliminated. Therefore, if any signaling at allcan be detected as a decreased 261-nt cassette/178-nt cassetteratio for wild-type (AATAAA) compared to mutant (TTTAAA) versions of pA�G178

catC9�, then that signaling cannot becleavage dependent.

Figure 7A shows that signaling, to the extent of 28% im-paired transcription in the wild type, was indeed detectedupon comparison of wild-type and mutant versions of thepA�G178

catC9� template. Although this is less signaling thanobtained with our other plasmids, we were nevertheless sur-prised at the vigor of the effect, since the poly(A) signal in thistemplate had been extensively mutated (all of its G residues).Despite the unusual sequence of the G-less poly(A) signal,there can be no doubt that the impaired transcription of thewild-type plasmid is a poly(A)-dependent effect because thetranscriptional impairment is abolished by mutating only twoof the A’s in the poly(A) hexamer (Fig. 7A). These resultsunambiguously establish that poly(A) signaling is cleavage in-dependent in vitro.

Although it was clear from the data discussed above that theobserved poly(A) signaling is cleavage independent, it re-mained possible that some processing at the G-less poly(A)site, unrelated to signaling, may also have occurred. If so the 5�polyadenylation product, and perhaps also the 3� cleavage andpartial degradation products, should be detectable on the gel(81). However, the lane containing the wild type in Fig. 7Areveals no evidence of any G-less poly(A) site processing.Poly(A) site cleavage in the 178-nt precassette would yield 5�28-nt and 3� 150-nt subcassettes. Polyadenylation of the 5�product would yield a heterogeneous collection of G-lessRNAs on the gel ranging from perhaps 180 to 280 nt. Partialdegradation of the 3� product would yield a heterogeneouscollection of G-less RNAs on the gel of less than 150 nt. Thereis no evidence in the wild type-containing lane of Fig. 7A forany of these products of processing. Thus, not only is signaling

cleavage independent in this assay but, at least for the G-lesspoly(A) site, there does not seem to be any processing at allunder these experimental conditions.

The G-less poly(A) site offers the advantage that any signal-ing detected by our assay must unambiguously have occurredby a cleavage-independent mechanism. However, the poly(A)site used for most of the experiments in the present study wasthe SV40 early poly(A) site. In order to determine whethersignaling is also cleavage independent for this site, we carriedout RNase protection assays on the products remaining aftersignaling reactions. Figure 7B shows that essentially no cleav-age at the SV40 early poly(A) site occurred under the condi-tions of our assays. Lane 2 shows the result of an RNaseprotection analysis of the in vitro transcription products of atypical 15-min signaling reaction. Lane 3 shows the result for asignaling reaction that was allowed to incubate three times aslong. Lane 4 shows the RNase protection result for authenticcellular mRNA containing the same poly(A) site. For the cel-lular RNA (lane 4) a band at the expected position for cleavedRNA (107 nt) is obtained, plus a subband. In contrast, for thein vitro RNA these bands are either scarcely visible (15-minreaction, lane 2) or only faintly apparent (45-min reaction, lane3). Instead, the in vitro RNA yields almost exclusively a pro-tected species migrating near the 129-nt position expected foruncleaved RNA. Additional bands flanking the position forcleaved RNA in lanes 2 and 3 are not related to 3� end for-mation as they appear also in assays of RNA from a plasmidbearing an unrelated poly(A) site (lane 1).

Quantitative analysis indicates an upper limit of less than2% poly(A) site cleavage of RNA transcribed in vitro for 15min (Fig. 7B, lane 2). In contrast, poly(A) signaling can lead toan elongation deficit of more than 60% under the same con-ditions (Fig. 1B). Thus, signaling has occurred in the virtualabsence of cleavage. Even after 45 min of incubation in vitrothe proportion of poly(A) site cleavage reaches only 6% (max-imum estimate from analysis of Fig. 7B, lane 3). We concludethat poly(A) signaling for the SV40 early poly(A) site has beenuncoupled from poly(A) site cleavage under our reaction con-ditions. Therefore, signaling is independent of cleavage forboth a natural and an artificial poly(A) site.

DISCUSSION

Poly(A) signaling in vitro. Poly(A)-dependent termination ispresumably a complex process in which poly(A) signaling is butthe first of several mechanistic steps. For example, signalingmay lead first to pausing and then to transcript release. Tofocus on signaling per se, therefore, we have used in this workan assay that does not require transcript release in order toproduce a measurable outcome. Rather, the assay detects anydownstream effect of signaling that impedes transcription.

Our results show that poly(A) signaling in vitro faithfullyreflects three important aspects of poly(A)-dependent termi-nation in vivo (79). First, of course, signaling is dependent ona functional poly(A) site. Second, signaling exerts its effectregardless of the nature of the downstream DNA: no specialpause site or other element is required. Third, the effect in-creases with distance, suggesting a stochastic process. Thus, thein vitro events characterized here recapitulate important as-


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pects of the in vivo process that leads to poly(A)-dependenttermination.

Detection of signaling under our assay conditions requirescitrate. The molecular target of citrate is not known, but citratehas been shown to inhibit phosphorylation of the RNAPIICTD (64) and is said to be an HIV-specific depressant ofelongation (63). Here we confirm, using an assay for elonga-tion efficiency, that citrate preferentially impairs elongation(Fig. 4A). Our results also show that the effect is not restrictedto HIV since our constructs are driven by the SV40 earlypromoter. Moreover, our elongation efficiency assay (Fig. 4)measures the efficiency of elongation between 895 and 2,757 bpdownstream of the promoter, showing that the effect of citrateis not limited to promoter-proximal events. Thus, the reductionof elongation efficiency imposed by citrate includes the regionof the template containing the poly(A) site. It is possible that,by reducing the phosphorylation of the CTD, citrate rendersthe polymerases more responsive to the poly(A) signal. Alter-natively, citrate may reduce a background of efficient elonga-tion contributed by nonresponding polymerases that otherwisewould mask signaling. It is also possible that citrate affectssignaling directly, by acting on some component of the tran-scription complex independently of its effect on elongationefficiency.

Using this poly(A) signaling assay we have shown here thatsignaling occurs well after extrusion of the poly(A) site, butbefore cleavage, and that it does not depend on the presence ofpause elements in the DNA. Assembly of the cleavage andpolyadenylation apparatus occurs in distinguishable steps invivo (18). The results reported here support our earlier sug-gestion (18) that signaling may occur during one of theseintermediate steps, being generated by new interactions amongknown factors (3, 8,14, 17) or by the recruitment of new factorsresponsible for termination.

Poly(A) signaling is not coupled to extrusion of the poly(A)site. Before 3�-end processing can occur, the poly(A) site mustfirst undergo extrusion and then stepwise assembly of thecleavage and polyadenylation apparatus (18). Cleavage-inde-pendent models for poly(A) signaling may accordingly be di-vided into intrinsic and extrinsic models according to whetherrecognition is by the transcriptional apparatus itself or by ad-ditional factors that recognize the signal following extrusion.Here we have addressed the intrinsic class of models whichpropose that the poly(A) site is recognized as it is being tran-scribed and extruded, thereby leading to termination. A pre-cedent for such a mechanism is E. coli RNA polymerase, whichcan recognize signals for pausing and termination either di-rectly in the DNA or in the nascent RNA during extrusion and

FIG. 7. Signaling is not cleavage dependent. (A) Signaling by aG-less poly(A) site is not accompanied by processing. The first step ofconstruction for pA�G178

catC9� was similar to that for pASw�C9377� ofFig. 3 except that a G-less version of S, the sequence shown in Fig. 7A,was inserted into pAPm�117cat�. The G of the blunted BamHI siteremaining after ligation was converted to a T by means of site-directedmutagenesis (AATCGATCC3AATattTCC), thereby fusing the G-less poly(A) signal with the 117-nt G-less cassette. To enhance thedetection of signaling, the distance between the cassettes was increasedby inserting the previously described C9 fragment into the uniqueEcoRV site (Fig. 4A) of the cat sequence. The cleavage site indicatedby the arrow in the wild-type G-less poly(A) sequence is the cleavageposition for the original SPA (5). A representative experiment isshown. Citrate was added with the nucleotides for these assays. (B)RNase protection analysis reveals negligible processing of the in vitrotranscription products. The same RNA probe was used for all samplesin the figure. This probe spans the identical poly(A) sites of pAPw�C9�

(lanes 2 and 3) and pIAPw�cat� (lane 4). The latter plasmid, used as apositive control, was obtained from the former by insertion of a PstI-RsaI fragment from pRL-SV40 (Promega) at the StuI site. This addsan intron to allow stable expression of RNA in vivo. The negativecontrol (lane 1) was pAL�117cat�, which has identical upstream se-quences to pAPw�C9� but a different poly(A) site. Note that the bandsmarked with asterisks are present irrespective of the presence or ab-sence of the homologous poly(A) site in the in vitro transcribed con-struct. The smudge in lane 1 at the position of uncleaved RNA is theedge of a halo from an intense band that was in the lane to the left onthe gel.


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whose responses to these signals can be modulated by factors(4, 58). Accordingly, in eukaryotes, RNAPII may recognize thepoly(A) site during transcription, and its response to thepoly(A) site may be modulated by the polymerase-associatedpoly(A) site cleavage factors that are known to be required fortermination (3, 8,14, 17). This model has never been tested butis now ruled out by the experiment of Fig. 6, which establishesthat signaling cannot have taken place during transcription ofthe poly(A) site, since all signaling can still be blocked byantisense after extrusion of the poly(A) site is complete.

Our data also address the simplest of the extrinsic modelsfor poly(A) signaling. This model, an extension of the abovescenario, is the CPSF-CstF recognition model, which proposesthat the signal to terminate is generated as the poly(A) site ispropelled past the CTD-bound CPSF and CstF after emergingfrom the polymerase (55). The initial interaction betweenthese factors and the poly(A) site is envisioned as the eventthat generates the signal. Both functional and structural con-siderations (see below) suggest that CPSF and CstF interactpromptly with the poly(A) site when it emerges from the poly-merase. Yet the results of Fig. 6 show that signaling has stillnot occurred 140 or more nt after extrusion is complete, de-spite the presumed efficiency with which the poly(A) signal iscaptured by CPSF and CstF. Accordingly, our results disfavorthe CPSF-CstF-recognition model and suggest that continuedassociation of CPSF and CstF with the poly(A) site is requiredin order to participate in generating the signal to terminate ata later step of cleavage and polyadenylation complex assembly.

Prompt targeting of CPSF and CstF to the poly(A) sitefollowing extrusion is suggested by analogy to the mechanismwhereby capping enzyme is targeted to the RNA 5� end. LikeCPSF and CstF, capping enzyme binds to the polymeraseCTD, which then delivers it to the emerging RNA (43, 54).This delivery is so efficient that the RNA is quantitativelycapped in vitro before it is 50 nt long (46). Additional studiesin vivo suggest that capping occurs when the RNA is between25 and 30 nt long (69). Since approximately 18 nt of RNA iscontained within the structure of the RNAPII ternary complex(39), capping must occur on 5� ends that lie only about 10 ntbeyond the point of extrusion. Thus, functionally, the CTDappears to be designed to deliver capping enzyme and possiblyother proteins to a region very close to the point at which theRNA emerges from the polymerase. This functional conclu-sion is consistent with the recently published crystal structureof RNAPII, which indicates that the newly extruded RNAemerges at the base of the CTD (25, 36).

Given the juxtaposition of the CTD to the emerging RNA,the presumed flexibility of the CTD (25), the role of the CTDin directing virtually immediate capping of emergent RNA,and the role of the CTD in facilitating 3� end processing, it islikely that the CPSF and the CstF on the CTD gain rapidaccess to the nascent poly(A) site. In this they would resemblethe vaccinia virus transcription termination factor, which alsotravels with the polymerase and then accesses its cognate ele-ment in the nascent RNA as soon as it emerges (40). Unlikethe vaccinia situation, however, initial recognition of thepoly(A) signal by CPSF and CstF does not appear to coincidewith the signal transduction event. Thus, some later event onthe way to processing is apparently responsible for signaling.Nevertheless, 140 nt of RNA would be insufficient to reach the

end of a hypothetical, fully extended, inflexible CTD. There-fore, the CPSF-CstF-recognition model remains a formal pos-sibility.

Poly(A) signaling is not cleavage dependent. Very soon afterit was discovered that termination is poly(A) dependent, it wassuggested that processing itself might be the signal that leads totermination (50). Subsequently, elegant models were devel-oped (20, 68) and refined (30, 81) to explain how cleavagecould be instrumental in generating the signal. Attempts to testcleavage-dependent models have centered on the relative tim-ing of cleavage and termination in vivo (11, 30, 61). The resultsfor several genes indicate that these events both occur at var-ious distances downstream of the poly(A) site but that, withinthe resolution of the experiments, they tend to occur together.This has led to the view that cleavage and termination in vivomay both be part of a single concerted event (11, 12, 30). It hasvariously been suggested that this event is triggered by cleavage(30) or that it is not triggered by cleavage (61). Both possibil-ities are consistent with the available in vivo data, as are theadditional possibilities that cleavage is triggered by terminationor that both occur independently in response to a commonsignal. Thus, the central postulate of the cleavage-dependentmodels, namely, that cleavage generates the signal to the poly-merase, has remained an open question.

As is often the case, the relationships among coupled eventsare clarified by uncoupling them in vitro. By uncoupling cleav-age and signaling in our assay we have shown that signalingoccurs in the essential absence of any cleavage. This was dem-onstrated directly for the SV40 early (Fig. 7B) and the G-lesspoly(A) sites (Fig. 7A). Moreover, because optimization of thesignaling assay involved selecting for conditions that suppressprocessing, this is evidently true for the SV40 late and thesynthetic poly(A) sites as well (Fig. 3). We hasten to point out,however, that our extracts do carry out efficient 3�-end pro-cessing under conventional cleavage and polyadenylation con-ditions (75) using exogenously prepared pre-mRNA substrates(data not shown).

Poly(A) signaling does not require downstream elements inthe DNA. We have shown here that four different poly(A) sitesare capable of signaling a stop to transcription in vitro in theabsence of any identifiable downstream elements. For exam-ple, the SV40 early poly(A) site transduces the signal to stoptranscription across both eukaryotic and artificial DNA se-quences (Fig. 5), as well as across prokaryotic DNA (Fig. 6A).The same is true for the SV40 late, the SPA, and the G-lesspoly(A) signals (Fig. 3, Fig. 7A, and additional data notshown). This confirms and extends our previous report thatpoly(A) signals can induce efficient termination in vivo withoutthe assistance of downstream elements in the DNA (79).

Nevertheless, a number of downstream elements have beendescribed that do enhance termination when located down-stream of poly(A) sites (2, 6, 13, 21, 31, 33, 72). However,only occasionally have the effects of the presence and absenceof these elements been determined in a background lackingpoly(A) signals (e.g., references 22, 31, and 41). Thus, often itis not known whether a poly(A)-independent effect has simplybeen superimposed on a poly(A)-containing background, orwhether such an element interacts synergistically with thepoly(A) site to constitute a true downstream member of abipartite terminator.


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Pause elements in downstream DNA are a prominent fea-ture of most discussions of poly(A)-dependent termination(e.g., see references 2, 6, 13, 33, 41, 73, 81, and 82). Theseelements are thought to be required to slow down the poly-merase so that the poly(A) signal can act. Yet, with one pos-sible exception, no termination-enhancing element has beenshown to pause polymerases in the absence of a functioningpoly(A) signal. For example, the nmt2 pause element ofSchizosaccharomyces pombe (2) does not induce pausing if thepoly(A) signal is deleted (41). Yet there are pausing and effi-cient termination downstream of the poly(A) signal if the el-ement itself is deleted (41). Thus, although the element isunquestionably an enhancer of polyadenylation (2), it is not apause element and it is not required for pausing or termina-tion. The possible exception referred to above is an elementthat was accompanied by both pausing and termination down-stream of a mutant poly(A) site in one experiment (33) butwhich gave no discernible pausing downstream of a wild-typepoly(A) site in other experiments (77).

If the various downstream elements that have been de-scribed are not pause elements and are generally not requiredfor termination, what might be their function? Since we haveshown both in vivo (79) and in vitro (this report) that a poly(A)signal alone is sufficient to stop transcription, these down-stream elements are unlikely to be an integral part of thepoly(A)-dependent termination mechanism. The so-called pause ele-ments discussed above, actually resemble enhancers or auxil-iary downstream elements for polyadenylation (19, 35, 52).They may function at the RNA level and affect termination bymodifying the pathway leading to cleavage and polyadenyla-tion itself. A second type of element functions at the DNAlevel to bind a protein that enhances processing and/or termi-nation when encountered by a transcription complex (6, 21,81). One such element, the MAZ protein binding site, en-hances cleavage and polyadenylation in vivo (6). MAZ boundto DNA causes pausing in vitro, but the pausing per se is notresponsible for the enhanced cleavage and polyadenylation,since pausing by other proteins has no effect on processingunless the polymerases are paused next to MAZ (80, 81). MAZmay, in fact, be a poly(A)-independent termination factor invivo (15), a property shared by the only other DNA-bindingprotein thought to be involved in termination by RNAPII (21).Significantly, both of these proteins are known primarily fortheir roles in transcription initiation. Thus, their roles in pro-cessing and termination may be primarily to act as fail-safedevices, not necessarily dependent on a poly(A) signal, de-signed to protect promoters from transcription interference(37).

ACKNOWLEDGMENTS

We thank Carol Eng in the laboratory of Arnie Berk for a constantsupply of HeLa cell starter cultures; Guillaume Chanfreau for insight-ful comments on the manuscript; and Ian Orozco, Amir Kazerouninia,and David Tsao for contributing clones.

This work was supported by NIH grant GM50863 and by an awardfrom the Jonsson Cancer Center Foundation.

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mechanism of poly(a) signal transduction to rna polymerase ii in vitro

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