Vol. 9, No. 11MOLECULAR AND CELLULAR BIOLOGY, Nov. 1989, P. 4986-49930270-7306/89/114986-08$02.00/0Copyright X) 1989, American Society for Microbiology

Unusual Enhancer Function in Yeast rRNA TranscriptionSTEWART P. JOHNSONt AND JONATHAN R. WARNER*

Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461

Received 5 May 1989/Accepted 2 August 1989

The rRNA genes in most eucaryotic organisms are present in a tandem array. There is substantial evidencethat transcription of one of these genes may not be independent of transcription of others. In particular, in theyeast Saccharomyces cerevisiae, the enhancer of rRNA transcription that lies 2.2 kilobases 5' of thetranscription initiation site is at least partly within the upstream transcription unit. To ask more directly aboutthe relationship of the tandemness of these genes to their transcription, we have constructed a minirepeatcontaining two identifiable test genes, with or without enhancer(s). On integration into the URA3 locus, thesegenes were transcribed by RNA polymerase I. A single enhancer effectively stimulated transcription of bothgenes by 10- to 30-fold, even when it was located upstream of both or downstream of both. Two enhancers hadroughly additive effects. These results suggest a model of enhancer function in tandemly repeated genes.

The transcription by RNA polymerase II of a wide varietyof genes is profoundly influenced by cis-acting short-nucle-otide sequences called enhancer elements that can lie up-stream or downstream of the transcription unit, at a distanceof up to several kilobases. In many cases, the enhancerelements bind protein factors that are necessary for en-hancer function. Although a number of models have beenproposed (reviewed in reference 30), a clear understandingof enhancer function is not at hand.Two features distinguish rRNA genes from other genes in

the eucaryotic cell. They are transcribed by a specificenzyme, RNA polymerase I, and they are arranged in atandem array. This unique genetic structure has led tospeculation that transcription of an individual gene is notindependent of transcription of the neighboring genes; e.g.,termination at one transcription unit may lead directly toinitiation at the neighboring downstream promoter (7, 12, 15,26).We have identified a sequence element, specific for RNA

polymerase I, that has many of the characteristics of anRNA polymerase II enhancer (9, 10). In this paper, weexplore aspects of the function ofRNA polymerase I and itsenhancer to ask about the parallels between polymerase Iand polymerase II enhancers and about the importance ofthe tandemness of rRNA genes to polymerase I activity.

Transcription by RNA polymerase I has been reviewedrecently (38). The spacers between individual rRNA tran-scription units vary in length from 2 kilobase pairs (kb)(yeasts) to >30 kb (mammals). Within them are found avariety of sequence elements that influence the level oftranscription. In Xenopus laevis, for example, these includeseveral copies of a sequence closely related to the promoter,dubbed spacer promoters (6, 7, 22, 23, 29, 33), each followedby about 10 copies of a 60- to 81-base-pair (bp) repeat, thatbehave in some respects like enhancer elements (22, 23, 29).The spacer promoters are curious because although they cangive rise to transcripts and can stimulate transcription froman adjacent real promoter (6, 7, 29), the transcription fromthe spacer promoter seems not to be involved in thisstimulation (7, 25). Repetitive elements, some related to thepromoter, have also been found in the nontranscribed spacer

* Corresponding author.t Present address: Department of Pathology, Duke University

Medical Center, Durham, NC 27710.

regions of the Drosophila (20, 31), mouse (21, 44), and rat (8)genomes. In the latter case, the sequence has some enhancerfunction (8).

Is the initiation of transcription of one gene in a tandemrepeat influenced by the termination of transcription of aneighboring gene? In both X. laevis (24) and Drosophilamelanogaster (42), there are reports that almost the entirespacer can be transcribed, with termination occurring a fewhundred nucleotides from the initiation site, although thisseems not to be a general phenomenon. In the mouse, arepetitive set of termination sequences (Sall boxes) havebeen identified about 600 bp downstream of the 3' end of the28S rRNA-coding region (13). Yet another Sail box is foundjust upstream of the initiation site; its presence stimulatestranscription substantially (12, 15). A termination sequenceupstream of the promoter stimulates transcription of a Xen-opus rRNA gene even when no upstream transcription unitis present (24). What is not clear is whether the terminationsequences immediately upstream of the promoter stimulatetranscription by recycling RNA polymerase I from one geneto the next (7, 28) or whether they are simply useful toprotect the promoter from readthrough by rogue polymerasemolecules that have escaped the upstream terminators (1,16) or that have initiated at a spacer promoter.

In Saccharomyces cerevisiae, the rRNA genes are ar-ranged as shown in Fig. 1A and B. The spacer regionincludes, surprisingly, the 5S RNA gene, but no repetitiveelements or promoterlike sequences are present (37). Oneshort region of 190 bp, an EcoRI-Hind III fragment (Fig. 1B),plays a particularly important role in rRNA transcription. Itincludes sequences that appear to be involved in terminationof transcription (18, 27), although downstream terminationsites come into play in the absence of the enhancer (18;unpublished results). It includes sequences that act as anRNA polymerase I promoter in vitro but probably not invivo (40, 41). Finally, it includes sequences that act as astrong enhancer of RNA polymerase I transcription at thenormal promoter (9, 10, 27).

This juxtaposition of termination and enhancer functionemphasizes the likely significance of the tandem nature ofthe ribosomal DNA (rDNA) genes in the transcription ofrRNA. Therefore, we have set up an experimental system totest various models of how termination, enhancement, andpromotion of transcription could be related. To do this, wehave constructed a mini-rDNA repeat containing two copies

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0.5kb "'URA3FIG. 1. The yeast rDNA repeat and constructs derived from it. (A) Two copies of the yeast rDNA repeat. Symbols: -., positions of the

35S pre-rRNA and 5S rRNA transcription units; 0, positions of the enhancer elements. (B) A single rDNA repeat unit, with the relevantrestriction enzyme cleavage sites indicated. The 190-bp EcoRI-HindIII enhancer element is indicated by a bolder line. (C) The T7 A-geneartificial rDNA cistron with an enhancer (for details, see reference 9). A 600-nucleotide segment from bacteriophage T7 has replacednucleotides 124 to 6085 of the 35S rRNA transcription unit. (D) The T7 B-gene artificial rDNA cistron with an enhancer (for details, seeMaterials and Methods). A 220-nucleotide segment from T7 has replaced nucleotides 44 to 6085 of the 35S rRNA transcription unit.Restriction sites: B, BamHI; Bg, BglII; E, EcoRl; H, HindIII; Pv, PvuII; 5, SinaI; Sa, SalI. Parentheses around a site indicates that it wasdestroyed during cloning. (E) Integrating plasmid YIP5 (---) containing the tandem repeat of the T17 A-gene artificial rDNA cistron (frompanel C; 8 o'clock to 12 o'clock) and the 177 B-gene artificial rDNA cistron (from panel D; 12 o'clock to 3 o'clock). This example has noenhancer elements. Enhancers can be inserted at the arrowheads (see Fig. 4). The ApaI site that is cut to direct integration at URA3 isindicated.

of the rDNA gene, in each of which most of the transcripthas been replaced by a distinct reporter sequence. Thisminirepeat was integrated into the URA3 gene, on a differentchromosome from the rDNA repeat. Under these condi-tions, the enhancer stimulates transcription ofboth upstreamand downstream genes and will even stimulate transcriptionof a distal gene. Two enhancers bracketing a gene have anadditive effect on transcription. The results are discussed interms of models of RNA polymerase I enhancer function,i.e., whether sliding or looping is involved, whether termi-nation is involved, and the role of tandemness.

MATERIALS AND METHODS

Strains and media. The host strain of S. cerevisiae isW303-1A (MATa ade2-1 leu2-3,112 his3-11 trpl-l ura3-1canl-100) (obtained from R. Rothstein, Columbia Universi-ty). W303-1A was grown at 30°C in synthetic completeminimal medium (45) with necessary supplements.Plasmid constructions. (i) B minigene. A 220-bp BamHI-

BglII fragment of bacteriophage T7 DNA (a generous gift ofJ. Dunn) was cloned into pGEM-3Z (Promega Biotec) at theBamHI site. The 590-bp EcoRl E fragment of rDNA, whichincludes the 3' terminus of 35S rRNA (32), was filled in withthe Klenow fragment ofDNA polymerase I and ligated to thefilled-in BamHI site of pGEM-T7B. The EcoRI B fragmentof rDNA, which spans the nontranscribed spacer from theenhancer fragment to 48 nucleotides beyond the initiationsite of 35S RNA (19), was ligated into the EcoRI site ofpGEM-T7B-EcoE. The downstream EcoRI site was de-stroyed by partially cleaving with EcoRI, filling in the ends,and religating the DNA. A BglII site was introduced adjacent

to the upstream EcoRI site by ligation of synthetic linkers.(Fig. 1D is a schematic representation of the T7B rRNAminigene.) This plasmid is referred to as pRR82'.

(ii) YIpRR88. pRR82' was linearized with HindIII and cutwith EcoRI, filled in, and ligated to BglII linkers (pRR83).This plasmid was cut with BglII and SalI, and the 3.0-kbfragment was ligated to BamHI-SalI-cut YIpRR10 (10).Schematic diagrams of this and all of the following plasmids(not including the YIp5 portion) are shown in Fig. 4.

(iii) YIpRR89. pRR82' was cleaved with BgIIl and SalI,and the 3.2-kb fragment was ligated to BamHI-Sall-cutYIpRR8 (10).

(iv) YIpRR92. pRR83 was cleaved within the polylinkerwith XbaI, filled in, and ligated to the EcoRI-HindIII en-hancer fragment, whose ends were also filled in (pRR85).This plasmid was cut with BglII and Sall, and the 3.2-kbfragment was ligated to BamHI-SalI-cut YIpRR8.

(v) YIpRR93. pRR83 was digested with BglII and Sail, andthe 3.0-kb fragment was ligated to BamHI-SalI-cut YIpRR8.(Fig. 1E).

(vi) YIpRR95. YIpRR88 was linearized with ClaI, filled in,and ligated to the EcoRI E fragment of rDNA, whose endswere also filled in. Transformants were screened by restric-tion digests to isolate a subclone whose insert was in theproper orientation.

(vii) YIpRR96. YIpRR92 was cut with SalI, filled in, andligated to the 750-bp HindIII-PvuII fragment isolated fromthe EcoRI B fragment. The orientation of the insert wasdetermined by restriction digestion.

(viii) YIpRR99. YIpRR95 was cut with SphI (which cutsonce within each of the two EcoRI B fragments), and the

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4988 JOHNSON AND WARNER

smaller SphI fragment was replaced with that fromYIpRR89.

(ix) YIpRR101. YIpRR96 was cut with NheI (which cutsthe plasmid twice, once within each of the two EcoRI Efragments), and the smaller NheI fragment was replacedwith that from YIpRR89.

(x) YIpRR102. YIpRR99 was cut with Sacl (which cuts inthe polylinker adjacent to the B reporter sequence) and NcoI(which cuts within the URA3 gene of the vector), and thesmaller fragment was replaced with the identical region fromYIpRR96.

(xi) YIpRR103. The small SphI fragment of YIpRR93 wasligated to the large SphI fragment of YIpRR102.

Transformation of yeast cells. Plasmid DNA (1 ,ug) isolatedfrom a dcm mutant strain of Escherichia coli (GM48) waslinearized with ApaI (Fig. 1E) (the ApaI site within theURA3 gene overlaps a recognition site for dcm methylase,and methylation appears to inhibit the restriction enzyme).In some instances, the NcoI site in the URA3 gene of theplasmid had been destroyed to generate a frameshift muta-tion to increase the frequency of integration at the URA3locus. Cells were grown overnight in rich medium to adensity of approximately 0.5 x 107 to 1.0 x 107 cells per ml.They were transformed with the linear DNA by the lithiumacetate method essentially as described by Ito et al. (17).

Isolation of yeast DNA. Total yeast DNA was isolated asdescribed previously (10). The DNAs were cut with HindIII,electrophoresed on 0.8% agarose gels, and blotted to nitro-cellulose (39). The filters were probed with -nick-translated,replicative form of bacteriophage fl DNA containing aninsert of the 1.1-kb HindIll fragment of the URA3 gene (34).RNA isolation and quantitation. RNA was extracted from

logarithmically growing cells (0.75 x 107 to 1.25 x 107/ml) asdescribed in method 2 of reference 9. Total cellular RNAs (5,ug) were separated on 1.5% agarose gels in 0.01 M NaPO4buffer (pH 6.5)-6% formaldehyde, blotted onto Nytran filters(0.45 ,um; Schleicher & Schuell, Inc., Keene, N.H.), andfixed by cross-linking with UV light (4). The filters wereprehybridized and hybridized in 5x SSPE (SSPE is 0.18 MNaCl, 0.01 M NaPO4, 0.01 M EDTA [pH 7.4)-5x Denhardtsolution-100 ,ug of salmon sperm DNA per ml-1% sodiumdodecyl sulfate-50% formamide at 55°C. Hybridizationprobes containing only phage sequence were antisense ribo-probes from the T7 bacteriophage DNA fragments clonedinto pSP64 (for T7A) or pGEM-3Z (for T7B). The probeswere synthesized by using [ot-32P]UTP (800 Ci/mmol; Amer-sham Corp., Arlington Heights, Ill.). Filters were washed in0.1 x SSPE at 650C. The amount ofRNA in each sample wasnormalized by reprobing the filter with a riboprobe of a300-bp EcoO109I-EcoRV fragment from the gene for ribo-somal protein L32 (5). An image analysis system (Quanti-met, Cambridge, United Kingdom) was used to scan auto-radiograms. The amount of RNA is expressed in arbitraryunits relative to the amount transcribed from YIpRR93. Theexperimental values are the means and standard errors of atleast three different Northern (RNA) blots.Run-on transcription. Run-on transcription was carried

out by permeabilizing cells with sodium N-lauryl sarcosineand subsequently incubating them with ATP, GTP, CTP,and [32P]UTP for 6 min at 250C as previously described (10).The radioactively labeled run-on transcripts were used tohybridize slot blot filters on which had been immobilizedcold antisense riboprobe RNAs for each of the respectivegenes. Nonradioactive probes complementary to T7 A- andB-gene sequences were made as described in the previousparagraph, substituting UTP for [a-32P]UTP. The sense and

antisense TCMJ riboprobes were from a subclone of the1.1-kb Xbal fragment of the TCMJ gene (35) cloned intopGEM-3Z. The enhancer probe was from the 190-bp EcoRI-HindlIl enhancer fragment cloned into pGEM-3Z. The tran-scribed spacer probe contained 112 nucleotides downstreamof the 18S rRNA sequences from a fragment of the rDNArepeat cloned into pGEM-3Z. Hybridization was carried outfor 38 h at 53°C in 50% formamide-5x SSPE-5x Denhardtsolution-200 ,ug of sonicated, denatured calf thymus DNAper ml-0.5% sodium dodecyl sulfate. The filters werewashed in 0.1 x SSPE-0.1% sodium dodecyl sulfate at 65°C.

RESULTSConstruction of a mini-rDNA repeat. Using an rDNA

minigene to analyze the effects on transcription of variousportions of the rDNA spacer, we found previously that a190-bp EcoRI-HindlIl fragment that is located 2.2 kb up-stream of the initiation site (Fig. 1) would stimulate rRNAtranscription approximately 15-fold (9). This stimulationoccurred with the fragment in either orientation as well aswhen it was placed downstream of the gene either on acircular plasmid or within a chromosome (10). By thesecriteria, this sequence is analogous to an RNA polymerase IIenhancer. However, two features distinguish it from mostenhancers described for genes transcribed by RNA polymer-ase II: it is located between identical transcription units, andit is at least partially transcribed as part of the end of theupstream transcription unit (18).To study how the enhancer may function in the tandem

array of the rDNA repeat, we constructed a test sequencecontaining two minigenes. Each contains the rDNA spacerregion including the promoter as well as the region of thegene that specifies the 3'-end formation of the precursorRNA (EcoRI E fragment) (Fig. 1C and D). Between thesetwo portions of the rRNA gene is a reporter sequence ofbacteriophage T7 DNA that replaces most of the rRNAsequences. The basic plasmid used for integration is shownin Fig. 1E. It can be modified to incorporate the EcoRI-HindIll enhancer fragment into the minirepeat either up-stream, between, downstream, or in various combinations.(Figure 1E; see also Fig. 4).The minirepeat was constructed on an integrating plasmid

so that the upstream and downstream positions of each genecould be studied unambiguously within the linear DNA of achromosome rather than on a circular plasmid. To havedirected the integration to the rDNA repeat would have beenself-defeating because the enhancer elements in adjacentgenes would have masked any effect of the element presentwithin the construct. Therefore, the integration was directedtoward the URA3 locus by transformation with plasmid cutwithin the URA3 gene. Analysis of the cellular DNA of Ura+transformants is shown in Fig. 2. The endogenous ura3 genewas found on a 1.1-kb Hindlll fragment (Fig. 2A, lane 1). Inthe construction of the integrating vector, YIpS, the HindIIIsites were destroyed so that integration at URA3 caused adisappearance of the 1.1-kb fragment and the appearance ofone or two new fragments (in this example, two; Fig. 2A,lane 2, and Fig. 2B). Integration frequently occurred withinthe rDNA repeat to yield an 8.2-kb fragment (Fig. 2A, lanes3 and 4). Occasionally, integration occurred at both sites(Fig. 2A, lane 3). For the experiments described in Fig. 4 and5, we identified transformants in which there had been asingle integration of the plasmid sequences into the URA3gene.

Transcription by RNA polymerase I. To determine whetherthe tandem genes inserted into the URA3 locus were both

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A9.4- * -Int. into rDNA6.6 -

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FIG. 2. Southern analysis of Ura+ transformants produced byintegration of YIpRR89. (A) HindIll-digested DNAs were electro-phoresed and transferred to Nytran filters, which were probed withURA3 sequences as described in Materials and Methods. Properintegration of the plasmid into the ura3-1 locus generated 5.0- and4.1-kb fragments (see Fig. 2B). Integration within the rDNA repeatgenerated a 8.2-kb fragment. The endogenous ura3-1 gene generateda 1.1-kb fragment. Positions of size markers (in kilobase pairs) areshown on the left. (B) Diagram of YIpRR89 (with an enhancerelement between the two T7 rRNA genes) integrated into URA3 toshow how one obtains 4.1- and 5-kb fragments on probing withURA3 sequences. Symbols: E, U, genomic and plasmid URA3sequences, respectively; El, rDNA sequences; 0, T7 A- and B-genesequences.

transcribed by RNA polymerase I, we assayed the sensitiv-ity of this transcription to oa-amanitin. Since yeast cells are

normally impermeable to ax-amanitin, the relative resistanceof transcription of each gene to a-amanitin was determinedby permeabilizing the cell membranes and then incubatingthe cells with [a-32P]UTP (10). The amount of RNA synthe-sized was measured by hybridization to an excess of an-tisense RNA bound to nitrocellulose. As shown previouslyfor the T7-A gene on a plasmid (10), the transcription of boththe T7 A and B genes was far more resistant to 166 ,ug ofa-amanitin per ml than was that of the ribosomal proteingenes TCMJ and RPL32 (Fig. 3). Raising the concentrationof a-amanitin to 400 ,ug/ml had little further effect (notshown). The transcription ofrRNA was similarly resistant toa-amanitin (lower bands). Thus, both T7 A- and B-generRNAs were transcribed by RNA polymerase I (36). Al-though we cannot formally rule out RNA polymerase III, itspromoter sequence requirements are altogether different.Furthermore, the T7 A-gene rRNA transcripts analyzedpreviously initiated precisely where 35S rRNA does (9).The probe used in the lower left slot in Fig. 3 was the

antisense strand from the 190-bp Eco-HindIII fragment thatwas the enhancer element. It is clear that it was beingtranscribed in the run-on assay to nearly the same degree aswas the spacer downstream of the 18S RNA sequences. Thisresult is further support for the conclusion that the enhancerelement is effectively transcribed in vivo (18).

FIG. 3. Run-on transcription of T7 A and T7 B genes. Run-ontranscription was carried out on cells in which YIpRR89 had beeninserted (see Fig. 4 and below) as described in Materials andMethods in the absence (A) or presence (B) of 166 ,ug of a-amantinper ml (10). Radioactive RNA was prepared and hybridized to filterson which had been immobilized RNAs complementary to the T7 Aand B genes, TCMJ, TCMJ (antisense), RPL32, and no RNA (B), as

diagrammed in panel C. A one-fifth sample of the labeled RNA was

hybridized to filters containing RNAs complementary to the EcoRI-HindlIl enhancer fragment (Enh) and to transcribed spacer (TS)sequences downstream of 18S rRNA. These filters were treated with0.4 ,ug of RNase A per ml before exposure to film. Althoughquantitation between genes is not very reliable in these run-on

assays, the comparison of intensity between T7 RNA (A or B) andrRNA is not unreasonable considering the fivefold difference ininput and the difference in probe length.

The enhancer element stimulates transcription of both genesof the minirepeat. In previous experiments, a single copy ofa minigene, with or without the enhancer fragment, wasintegrated at the URA3 locus (10). The enhancer stimulatedtranscription four- to fivefold.We next examined the effects of the enhancer on a

minirepeat in this same locus. Integration of the constructthat lacked the enhancer fragment (YIpRR93; Fig. 4) led totranscription of both genes at a low level (Fig. 5). This was

approximately the same level of transcription observed foreach when it was integrated as a single-copy minigene (datanot shown). The presence of tandem copies of the repeat didnot lead to a substantial increase in transcription.

In the ribosomal repeat, there is an enhancer elementbetween each transcription unit. When this situation was

duplicated in the minirepeat construct (YIpRR89), i.e., anenhancer fragment was placed between the two minigenes,there was a marked increase in the transcription of each gene(Fig. 5), approximately 26-fold for the upstream A gene andapproximately 16-fold for the downstream B gene (Fig. 4).The result for the A gene is particularly striking, since in S.cerevisiae an element affecting transcription by RNA poly-merase II does not function when placed downstream of itstarget gene (14).

Thus, the enhancer is even more effective within theminirepeat than when integrated upstream or downstreamwith a single minigene (10). Furthermore, a single enhanceris able to stimulate transcription of both upstream anddownstream genes. It is interesting that integration of theenhancerless YIpRR93 into the rDNA repeat, rather thaninto the URA3 locus, led to approximately 50-fold-moretranscription of both A and B genes, presumably due toadjacent enhancer sequences (data not shown).The enhancer stimulates transcription of a distal promoter.

As shown above, the enhancer is able to affect the transcrip-tion of both genes when it is located between them, showing

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4990 JOHNSON AND WARNER

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FIG. 4. Effect of the enhancer on transcription of the T7 A- and B-gene cistrons of the rDNA minirepeat. A schematic drawing of eachof the constructs is presented, along with the relative amounts of T7 A- and B-gene rRNAs. The numbers shown were determined as describedin Materials and Methods. In each diagram (not drawn to scale), the long open box represents the HindIII-EcoRl fragment of the spacer regionof the rDNA repeat, the short open box is the HindIII-PvuII fragment of the spacer region, the box with a T is the EcoRI E fragment of therDNA repeat that contains the sequences necessary for 3'-end formation of the pre-rRNA, the box with the arrowhead is the EcoRI-HindIIIenhancer element, the hatched box represents the T7 A-gene sequences, and the closed box is the T7 B-gene sequences.

little if any bias toward either promoter. If the enhancer werefunctioning by binding a transcription factor or RNA poly-merase I which then could shuttle linearly along the DNAuntil it located the promoter, then it should stimulate tran-scription of the proximal promoter when it is placed eitherupstream or downstream of both genes. To investigate thispossibility, we made constructs with an enhancer eitherupstream (YIpRR88) or downstream (YIpRR92) of the mini-repeat.Using plasmid YIpRR88, there was stimulation of tran-

scription of both the A and B genes (Fig. 5). The enhancerwas more effective on the proximal promoter (A gene) butstill stimulated transcription of the distal (B) gene by fivefold(Fig. 4). It is striking that the enhancer could affect transcrip-tion of the B gene in this construct even though it lies 5.5 kbupstream of the gene and is separated from it by the T7A-gene rRNA transcription unit.When the enhancer was placed downstream of the minire-

peat as in YIpRR92, there was again stimulation of transcrip-tion of both genes, greater for the proximal one (Fig. 5).Nevertheless, the enhancer effected a fivefold stimulation of

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Total RNA was isolated from Ura3+ transformants of each of theindicated plasmids, electrophoresed, transferred to Nytran filters,and probed as described in Materials and Methods. A single blot wasprobed consecutively with the different probes. In this print, someof the RNAs have been overexposed to allow visualization of theRNAs in YIpRR93.

the distal A gene, located at a distance of more than 4.0 kbupstream.The level of stimulation observed in these two constructs

was lower than that measured when the enhancer waslocated between the two genes. Since enhancer function canbe partially or totally eliminated by foreign flanking se-quences (10), we modified YIpRR88 and YIpRR92 to im-merse the enhancer in its natural environment. In the result-ing constructs, YIpRR95 and YIpRR96 (Fig. 5), theenhancer stimulated transcription of each gene approxi-mately twice as much as in YIpRR88 and YIpRR92 (Fig. 4).In each of these transcripts, the level of transcription of theproximal gene was now approximately that of YIpRR89,suggesting that the enhancer functions most effectively whenpresent in its native environment.

Multiple enhancers have an additive effect on transcription.In the normal tandem array of the rDNA repeat, each genehas both an upstream and a downstream enhancer that itshares with its upstream and downstream neighbors. Todetermine the effects of an enhancer in each of the tandemrepeats, we tested a number of constructs containing two orthree enhancer elements. Note that adding a second en-hancer in tandem within one repeat is actually inhibitory(10).

In YIpRR99, the two enhancers, one upstream of theminirepeat and the other between the two minigenes, led toa dramatic stimulation in transcription of both the A gene(approximately 80-fold) and the B gene (approximately 30-fold) (Fig. 4 and 5). Quantitatively, this degree of stimulationis approximately the arithmetic sum of that observed in thesingle-enhancer constructs, YIpRR89 and YIpRR95. A sim-ilar result was observed when enhancers were located bothbetween and downstream of the two genes (YIpRR101) (Fig.4 and 5). On the other hand, when the two enhancers werenot in adjacent repeats (YIpRR103; Fig. 4 and 5), theireffects were not additive. In this instance, the assay may notbe sufficiently sensitive to measure the smaller effect that thedistal enhancer has on each gene.A final minirepeat was constructed with an enhancer

present in all three locations (YIpRR102). The three enhanc-ers were only marginally more effective than two adjacentenhancers (Fig. 4 and 5). Perhaps two enhancers are suffi-cient to effect transcription of the genes at a maximal rate.

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DISCUSSION

We have asked how the RNA polymerase I enhancer of S.cerevisiae may function in vivo by constructing a tandemrepeat of two mini-rDNA genes. When placed between thetwo genes, the enhancer stimulates transcription of both.When placed at either end of the repeat, the enhancer againstimulates transcription of both, although there is a greatereffect upon the proximal promoter. Also, if enhancers areplaced at both ends of a minigene within the repeat, theincrease in the rate of transcription approximates the sum ofthat due to two single-enhancer units.One possible explanation of these results is that in some

cells or at some times the enhancer can loop to associatewith the T7 A gene and in others with the 17 B gene. Thisdoes not occur with enhancers of polymerase II transcription(30) but is difficult to rule out experimentally in our tandemcontruct. In any case, this possibility has little impact on themodel presented below.

It is useful to compare the enhancer described above withthe 60- to 81-bp repeats that have enhancer activity forrRNA transcription in Xenopus oocytes and which also workfrom upstream of two genes or between two genes (22, 23).The two enhancers differ in several respects. The effect ofthe Xenopus enhancer, at least in oocyte injection experi-ments, is apparent only in a competition assay, where itreduces transcription of enhancerless genes in trans withoutsignificantly affecting transcription of genes in cis (22). TheSaccharomyces enhancer stimulates transcription of geneswith which it is' associated (9; Fig. 4). Furthermore, unlikethe Saccharomyces enhancer, the Xenopus enhancer hasmultiple regions of extensive sequence homology with a siteon the promoter involved in binding RNA polymerase I andthus is likely to be sequestering polymerase molecules,transcription factors, or both.The mechanism of action of the enhancer remains unclear.

One laboratory has suggested that it may function as aspacer promoter, since it was found to be a promoter in vitro(40, 41). However, no such spacer promoter has beenobserved in vivo in S. cerevisiae. Our results are inconsis-tent with the enhancer acting as a spacer promoter, since adownstream enhancer is nearly as effective as an upstreamenhancer (Fig. 4).Another possible mechanism for the enhancer involves the

role that termination may play in the transcription of tan-demly arranged genes. In both mouse and Xenopus nucleoli,there is evidence that termination of upstream transcriptionis linked to initiation at the downstream promoter (7, 12, 15,26). This could increase the efficiency of transcription byhanding the RNA polymerase I from one gene to the next insome way (7, 28), since the polymerase molecule would notdissociate from the tandem repeat. However, recent evi-dence from studies on Xenopus transcription casts doubt onthe handover model (25). In this fashion, the polymerasemolecule would not dissociate from the tandem repeat. Inyeast cells, transcription also extends into the spacer. All ofthe putative termination sites lie upstream of the 5S gene;one of these sites has been localized to the enhancer (18, 27).Thus, the enhancer could function by capturing terminatingRNA polymerase I molecules and in some manner passingthem to a promoter. However, our results are incompatiblewith this mechanism. The enhancer in YIpRR95, which isupstream of all RNA polymerase I transcription units, is aseffective as one which is downstream (YIpRR89 orYIpRR96).RNA polymerase II enhancers have been proposed to

35S Pol I_ 53V ".x'0~~-l::...' i'T/E.j

53~~5

FIG. 6. Model of the association of the ends of the rRNAtranscription unit. The model postulates that there is a physicalassociation of the terminator-enhancer elements (T/E) with thepromoter elements (P). For clarity, and to emphasize that manyproteins are involved as well as the DNA sequences, the sizes ofthese elements have been magnified compared with the length of the35S transcription loop or the 5S transcription loop. Three repeatsare shown, although many more could be associated in a three-dimensional structure. In practice, the structure would probablyhave less rigid valence and symmetry than is indicated by thedrawing. We suggest that on termination within or around a termi-nator-enhancer element, the RNA polymerase I would be availableto, and adjacent to, many promoter elements. The high localconcentrations of polymerase and promoter, and presumably tran-scription initiation factors, would lead to effective utilization of thepolymerase for reinitiation.

function by many mechanisms; two of the most prominentare scanning and looping (for a review, see reference 30). Inthe scanning model, the enhancer is a high-affinity bindingsite for RNA polymerase (or, more likely, one or severaltranscription factors). After binding, the polymerase diffusesunidimensionally along the DNA until it identifies a pro-moter, whereupon it initiates transcription. In the loopingmodel, the enhancer interacts with the promoter by means ofproteins bound to the DNA, thus looping out the interveningDNA.These models of enhancer function have been adapted for

the tandem repeat of rDNA genes. One model is linear, inwhich enhancer-binding proteins shuttle the polymeraseupstream or down, scanning for the next promoter (11). Theother is looped, in which there is a direct interaction betweenthe terminator and promoter of a gene. In this view, apolymerase molecule would transcribe again and againthrough a single gene (18).Our results support neither model. For example, in

YIpRR95 and YIpRR96, where the enhancer is upstream ordownstream of both genes, there is substantial transcriptionfrom the distal promoter. If an RNA polymerase I moleculeis moving from the enhancer to the promoter as proposed inthe scanning model (11), then there should be little if anytranscription from the distal promoter. On the other hand, asimple model suggesting looping between terminator andpromoter (18) does not explain how a terminator-enhancer

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4992 JOHNSON AND WARNER

can stimulate transcription from a downstream gene, nordoes it explain how it stimulates transcription of a distalgene, upstream or downstream.An alternative (Fig. 6) expands the loop model into three

dimensions. One could envision a loosely organized struc-ture where several (or all) of the terminator-enhancer ele-ments and the promoter elements are juxtaposed. The 35SRNA and the 5S RNA transcription units would loop outfrom this central structure, as suggested by a lampbrushchromosome (3). The enhancers would facilitate the passageof RNA polymerase I, or a transcription complex includingpolymerase and associated transcription factors, from theend of one transcription unit to the beginning of any other,either by direct interaction or simply by generating a localhigh concentration of polymerase molecules. An advantageof this model for explaining our results is that any particularenhancer can join the complex whether or not it has itselfbeen transcribed. Furthermore, the model explains how anenhancer could stimulate transcription of a distal gene. Inpractice, the assembly of enhancers and promoters wouldpresumably have less rigid valence and symmetry thanshown in Fig. 6, explaining why there is less enhancement ofa distal gene. One could imagine that the sensitivity oftranscription by RNA polymerase I to inhibition of topo-isomerase activity (2) is due to the disruption of the enhanc-er-promoter structure by hypersupercoiling.The model also takes into account the presence of the 5S

RNA genes in the spacers between the major rRNA genes(Fig. 1A). On the one hand, the presence of the 5S RNAtranscription unit does not interfere with the function of thepolymerase I enhancer. On the other, the enhancer does notaffect 5S RNA transcription (L. Neigeborn and J. R. War-ner, manuscript in preparation), since the 5S genes aredistinct from the enhancer structure. The model furthersuggests that additional sequences in the 5S RNA loop, evenwhole genes, might not interfere with the effect of theenhancer on a downstream gene.To stabilize such a structure, there must be elements,

presumably proteins but possibly small RNAs as well (43),that maintain the association between the promoters and theenhancers. We have identified one candidate protein, termedREB1, that binds to a specific sequence present within boththe enhancer and promoter (28a). There are likely to beothers. The model predicts a physical association betweenrRNA transcription units tens or hundreds of kilobasesapart. This would provide a mechanism for concentratingtranscription initiation factors and RNA polymerase I at thetranscription initiation site in the interests of maintaining amaximal level of transcription.

ACKNOWLEDGMENTS

This work was supported by Public Health Service grantsGM25532 and CA13330 from the National Institutes of Health andby American Cancer Society grant MV-323S.We are grateful to Mary Studeny, who provided tireless, dedi-

cated technical assistance, to Qida Ju and Bernice Morrow foruseful discussions, and to Julius Marmur, Lorraine Marsh, and IanWillis for constructive comments on the manuscript.

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