cis- and trans-Acting Determinants of Transcription Termination by Yeast RNA Polymerase II

Most eukaryotic genes are transcribed by RNA polymerase II (PolII), including those that produce mRNAs and many noncoding functionalRNAs. Proper expression of these genes requires efficient terminationby Pol II to avoid transcriptional interference and synthesisof extended, nonfunctional RNAs. We previously described a pathwayfor yeast Pol II termination that involves recognition of anelement in the nascent transcript by the essential RNA-bindingprotein Nrd1. The Nrd1-dependent pathway appears to be usedprimarily for nonpolyadenylated transcripts, such as the smallnuclear and small nucleolar RNAs (snoRNAs). mRNAs are thoughtto use a distinct pathway that is coupled to cleavage and polyadenylationof the transcript. Here we show that the terminator elementsfor two yeast snoRNA genes also direct polyadenylated 3'-endformation in the context of an mRNA 3' untranslated region.A selection for cis-acting terminator readthrough mutationsidentified conserved features of these elements, some of whichare similar to cleavage and polyadenylation signals. A selectionfor trans-acting mutations that induce readthrough of both asnoRNA and an mRNA terminator yielded mutations in the Rpb3and Rpb11 subunits of Pol II that define a remarkably discretesurface on the trailing end of the enzyme. Our results suggestthat, at least in budding yeast, protein-coding and noncodingPol II-transcribed genes use similar mechanisms to direct terminationand that the termination signal is transduced through the Rpb3/Rpb11heterodimer.

INTRODUCTION

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Introduction

A typical eukaryotic chromosome contains hundreds or thousandsof genes arrayed along both strands of the DNA. Most of thesegenes are monocistronic, i.e., correspond to a transcriptionunit, so each must include transcription start (promoter) andstop (terminator) signals. In the yeast Saccharomyces cerevisiae,the distance between transcription units is often only a fewhundred nucleotides or less, so, depending on gene orientation,promoters and terminators are adjacent or even overlapping.Such a genomic arrangement puts stringent demands on the efficiencyof transcription termination, since readthrough is expectedto result in occlusion of the downstream promoter if the genesare in the same orientation and formation of antisense RNA and/orcolliding polymerases if the genes are in the opposite orientation.Either outcome would likely result in aberrant gene expression(36).

Most eukaryotic genes are transcribed by RNA polymerase II (PolII) (23). Termination of transcription by Pol II is coupledto formation of the 3' end of the transcript (4, 35), whichoccurs by different mechanisms for protein-coding and noncodingRNAs. The 3' ends of mRNAs are formed by cotranscriptional endonucleolyticcleavage, followed by polyadenylation of the 3' end thus generated(49). The cis-acting elements that direct cleavage and polyadenylationin yeast are quite degenerate (22), but statistical analysisof a large number of known and putative processing sites hasresulted in algorithms for the probabilistic prediction of mRNA3' ends (20). Soon after transcript cleavage, termination oftranscription and degradation of the 3' fragment of the cleavedtranscript occurs, although the temporal order and interdependenceof these two events are uncertain.

Formation of the 3' ends of small nuclear RNAs (snRNAs) andsmall nucleolar RNAs (snoRNAs) differs between vertebrates andyeast. In vertebrates, most snoRNAs are processed from pre-mRNAintrons (18), while snRNA genes utilize specialized promotersand 3'-end formation elements distinct from those that directmRNA synthesis (24, 32). In contrast, all yeast snRNAs and mostyeast snoRNAs are synthesized autonomously from promoters thatappear similar to mRNA promoters. The 3' ends of these RNAsare generated by either endonucleolytic cleavage or termination,followed by trimming by the nuclear exosome, a complex of 3'exonucleases (6). In cases where endonucleolytic cleavage ofsnRNAs or snoRNAs is known to occur, it is not clear if thiscleavage is coupled to termination.

We recently reported a mutational analysis of the terminatorfor a yeast snoRNA gene, SNR13 (39). The SNR13 terminator consistsof at least two elements (regions I and II), which togetherspan about 100 nucleotides. Here we place the SNR13 terminatorin the 3' untranslated region (UTR) of a protein-coding geneand find that, surprisingly, it directs polyadenylation of themRNA. The polyadenylation sites map immediately downstream ofregion II, which contains sequences similar to mRNA cleavageand polyadenylation elements. A parallel analysis of the SNR65snoRNA terminator yields similar results, suggesting that bipartiteterminators containing cleavage and polyadenylation signalsmay be a common characteristic of yeast snoRNA genes. Usingboth the SNR13 snoRNA terminator and the CYC1 mRNA terminator,we selected for trans-acting mutations that result in terminatorreadthrough and obtained substitutions in the Rpb3 and Rpb11subunits of Pol II. These subunits form a heterodimer that ishomologous to the ${alpha}$ subunit homodimer of bacterial RNA polymeraseand is stabilized by a long coiled-coil interaction. The readthroughmutations cluster at the extreme C terminus of the Rpb11 dimerizationhelix and an interacting residue in Rpb3. We propose that thissurface is a contact point for factors that transmit the terminationsignal to Pol II.

MATERIALS AND METHODS

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Materials and Methods

Plasmids. pGAC24 contains the ACT/CUP fusion gene, including CUP1 3' UTRsequences, between the TDH3 promoter and the PGK1 3' UTR andpolyadenylation site (31). pGAC24 ${Delta}$ UTR lacks the CUP1 3' UTR butretains the PGK1 3' UTR and was created by replacing a 751-bpBamHI-SalI fragment of pGAC24 with a 594-bp BamHI-SalI fragmentof a PCR product made with the 5'-CUP and CUP1-3'-SalI primers.The 217-bp SNR65/RPS14A intergenic region was amplified by PCRwith flanking XhoI sites with primers SNR65-XHO-101F and SNR65-XHO-317Rand inserted into the intronic XhoI site of pGAC24 to createpGAC24-SNR65F and pGAC24-SNR65R or into the SalI site of pGAC24- ${Delta}$ UTRto create pGAC24-65UTR-F and pGAC24-65UTR-R. A single-nucleotidesubstitution of C for T is encoded within the SNR65-XHO-101Fprimer relative to the sequence of the SNR65/RPS14A intergenicregion currently deposited in the Saccharomyces Genome Database(www.yeastgenome.org/) due to an update in the database occurringafter the primers were created. Mutational analysis and mappingof polyadenylation sites were carried out with constructs havingthis variant sequence. A primer with a sequence matching thecurrent database entry, SNR65-XHO-101F-T, was subsequently usedto create versions of pGAC24-SNR65F and pGAC24-SNR65R that perfectlymatch the SGD sequence. No difference in copper sensitivitywas observed with these corrected constructs compared to thevariants. The corrected SNR65-XHO101F-T primer was used withSNR65-XHO-236R to create pGAC24-SNR65-101-236.

RNA analysis. Northern blots were prepared as described previously (39), with20 µg of total cellular RNA per lane, and probed with³²P-labeled oligonucleotide 3'CUP-2 or ACT1-probe. To map sitesof polyadenylation, 0.1 to 1 µg of total cellular RNAprepared by the glass bead-guanidinium isothiocyanate-hot phenolmethod (45) was combined with 250 pmol of T16-BSG1 primer in26.5 µl of H₂O, incubated at 95°C for 10 min and 65°Cfor 10 min, and cooled on ice. Reaction mixtures were broughtto 50 µl by the addition of 5 µl of 100 mM dithiothreitol,5 µl of deoxynucleoside triphosphates (10 mM each), 1µl of RNasin (Promega), 10 µl of 5x avian myeloblastosisvirus reverse transcriptase buffer, and 2.5 µl of avianmyeloblastosis virus reverse transcriptase (USB) and incubatedat 42°C for 1 h. One-fifth of the reverse transcription(RT) reaction mixture was used directly in PCRs with the T16Bsg1and 5'-CUP primers and Tfl DNA polymerase (Epicenter Technologies).Products were digested with BamHI and SalI, cloned into pUC118,and sequenced with M13 universal primers.

Isolation of cis-acting readthrough mutations. Error-prone PCR of the ACT-CUP intron region of pGAC24-SNR65F,cotransformation of PCR products with gapped pGAC24, and selectionof copper-resistant colonies were carried out as described previously(39).

Isolation of trans-acting readthrough mutations. The genome-wide selection for spontaneous SNR13 terminator readthroughmutations was described previously (39). One mutant (from the46a background; MATa cup1 ${Delta}$ ura3 his3 trp1 lys2 ade2 leu2) havinga cold-sensitive growth phenotype was transformed with a YCp50-basedyeast genomic library to identify clones that enable growthat 16°C. A single complementing clone with a genomic insertof $~$ 14.4 kb containing RPB11 and several other genes was identified,and subcloning revealed that the complementing activity wascontained within a 1.55-kb EcoRI-SacI fragment containing onlyRPB11 and the extreme 5' end of SIN3. Sequencing of the genomicRPB11 locus in the mutant strain revealed a GAG-to-GGG mutationin codon 108.

DNA fragments containing wild-type RPB3, RPB7, and RPB11 alleleswere amplified from yeast strain 46 ${alpha}$ (isogenic with 46a exceptMAT ${alpha}$ ) with KOD Hi-Fi DNA polymerase (Novagen) and cloned intoURA3-marked centromere plasmid pRS316. Mutagenesis was carriedout by PCR of these plasmid clones with Tfl DNA polymerase (Epicenter)with universal M13 forward and reverse primers and deoxynucleosidetriphosphates at 200 µM each. The resultant PCR productswere cotransformed with EcoRI/BamHI-digested pRS316 plasmidDNA into yeast strain 46 ${alpha}$ harboring LEU2-marked reporter plasmidpGAC24-SNR13 (40) or pGAC24-CYC83F (39). Transformants wereselected on medium lacking uracil and leucine, and colonieswere then replica plated onto medium lacking leucine and containing0.15 or 0.25 mM CuSO₄ to select for terminator readthrough.To test for linkage of the copper-resistant growth phenotypeto the mutagenized plasmid, strains were streaked onto platescontaining 5-fluoroorotic acid and lacking leucine to selectfor loss of the URA3-marked plasmid and then scored for lossof copper resistance.

Plasmid-borne Pol II subunit genes from copper-resistant strainswere amplified with KOD Hi-Fi DNA polymerase with M13 universalprimers and sequenced with a combination of M13 and gene-specificprimers. PCR products from selected RPB11 mutants (see Table2) were cloned into TRP1-marked centromere plasmid pRS314 toconfirm linkage of the copper resistance phenotype and to testfor function in the absence of wild-type RPB11. The genomicRPB11 locus of 46 ${alpha}$ bearing a second copy of RPB11 on URA3-markedcentromere plasmid pRS316 was disrupted by transformation withan rpb11 ${Delta}$ ::KanMX4 fragment amplified from an rpb11 disruptionstrain obtained from Research Genetics and selection for G418resistance. Integration at the RPB11 locus was confirmed byPCR and by dependence on a plasmid-borne RPB11 allele for viability.Mutant rpb11 alleles in pRS314 were transformed into 46 ${alpha}$ ${Delta}$ rpb11,tested for terminator readthrough activity by plating on mediumcontaining copper, and tested for function in the absence ofRPB11 by plating on medium containing 5-fluoroorotic acid. Allof the mutant alleles tested (see Table 2) conferred resistanceto at least 0.15 mM copper.

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TABLE 2. Dominant terminator readthrough mutations in RPB11 and RPB3

Oligodeoxynucleotides. The oligodeoxynucleotides used were as follows: 5'-CUP, 5'-CAAGAACTTAGTTTCGACGG-3';CUP1-3'-SalI, 5'-GACTAGTCGACTTATTTCCCAGAGCAGCA-3'; 3'CUP-2,5'-ACTCATGACCTTCATTTTGGAAC-3'; T16BSG1, 5'-GCGTCGACGAATTCGTGCAGTTTTTTTTTTTTTTTTV-3'(V = A-C-G mixture); SNR65-XHO-101F, 5'-TCCGCTCGAGCGATTTTGCAACTCTTGGTA-3';SNR65-XHO-101F-T, 5'-TCCGCTCGAGCGATTTTGTAACTCTTGGTA-3'; SNR65-XHO-317R,5'-TCCGCTCGAGGTTATGCATGTATTGTACTT-3'; SNR65-XHO-236R, 5'-TCCGCTCGAGATCAAATCAGCTCATACGTC-3';ACT1-probe, 5'-TCAGTAAATTTTCGATCTTGGGAAG-3'. Underlining indicatesthe single-nucleotide substitution of C for T that is encodedwithin the SNR65-XHO-101F primer relative to the sequence ofthe SNR65/RPS14A intergenic region currently deposited in theSaccharomyces Genome Database (www.yeastgenome.org/).

RESULTS

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Results

Characterization of the SNR65 terminator. We previously mapped the SNR13 snoRNA gene terminator by insertingPCR-mutagenized SNR13 downstream sequences into the intron ofthe ACT/CUP fusion gene (31) and selecting for mutations thatallow readthrough of the terminator by demanding growth in thepresence of >0.1 mM copper (39) (Fig. 1). The SNR13 terminatorwas found to be bipartite, consisting of two $~$ 50-bp sequencesdesignated region I and region II. Region I contains sequencesexpected to bind Nrd1, while region II contains features similarto pre-mRNA cleavage-polyadenylation sites. Each region hasweak terminator activity, but both are required for efficienttermination and point mutations in both are required for strongreadthrough (39).

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FIG. 1. (A) Locations of terminator elements downstream of the SNR13 and SNR65 snoRNA genes. Numbers indicate position in base pairs from the transcription start site of the snoRNA genes. Boxes with heavy outlines indicate either the coding sequence for the mature snoRNAs snR13 and snR65 or the open reading frames for the downstream protein-coding genes. Arrows indicate the directions of transcription. Boxes with thin outlines demarcate the region I and region II terminator elements of SNR13 (39) or SNR65 (this study). (B) Scheme for selection of cis-acting mutations in snoRNA terminators (term.). See text for details. (C) Copper plate assay for SNR65 terminator function. Tenfold serial dilutions of cultures of the 46 ${alpha}$ cup1 ${Delta}$ (WT [wild type]) and nrd1-5 mutant strains harboring ACT/CUP reporter plasmids with no insert or with the indicated SNR65 3'-flanking sequences (SNR65long, bp 101 to 317; SNR65short, bp 101 to 236) inserted into the intron were spotted onto medium lacking leucine and containing the indicated concentrations of copper and incubated for 2 to 3 days at 30°C. (D) Northern analysis of ACT/CUP RNA. Total cellular RNA was prepared from the 46 ${alpha}$ (WT) and nrd1-5 mutant strains harboring ACT/CUP reporter plasmids with no insert or with SNR65long (L) or SNR65short (S) sequences inserted into the intron. The blot was probed with ³²P-labeled oligonucleotide ACT1-probe, which is complementary to the ACT1 mRNA 5' UTR and so detects ACT/CUP mRNA, as well as endogenous ACT1 mRNA.

To search for elements common to snoRNA terminators, we subjectedthe SNR65 downstream region to the same analysis. We chose SNR65both because it has a small intergenic interval of only 217bp and, unlike SNR13, it is convergent with the downstream gene(RPS14A, Fig. 1A). Thus, the SNR65 terminator is adjacent toor interdigitated with a cleavage-and-polyadenylation site onthe opposite strand. This arrangement may demand a terminatorstructure different from that of SNR13, which is transcribedin the same direction as its downstream gene.

It was anticipated that insertion of the 217-bp SNR65/RPS14Aintergenic fragment into the ACT/CUP intron in either orientationwould result in decreased expression of the ACT/CUP gene, dueto either the SNR65 terminator in the forward orientation orthe RPS14A cleavage-and-polyadenylation site in the reverseorientation. Surprisingly, while the forward orientation conferredsevere copper sensitivity (SNR65long, Fig. 1C), the reverseorientation did not (data not shown). Thus, the RPS14A cleavage-and-polyadenylationsite does not appear to function in the context of the ACT/CUPintron.

We examined the role of the Nrd1 protein in SNR65 terminatorfunction by testing copper sensitivity in a nrd1-5 mutant strain(Fig. 1C). No appreciable increase in copper resistance wasimparted by the nrd1-5 mutation with the SNR65long insertion,suggesting that Nrd1 may not be required for SNR65 terminatorfunction. However, sequence alignment of the SNR65 3'-flankingregion revealed two blocks of similarity with the SNR13 terminator(Fig. 2), corresponding roughly to regions I and II but separatedby a spacer region of about 50 nucleotides. Furthermore, regionI of SNR65 is highly similar to a region of the NRD1 5' UTRthat is required for negative autoregulation of Nrd1 level bypremature transcription termination (40) (Fig. 2). Therefore,we tested whether a smaller segment of SNR65 3'-flanking sequencecontaining the region I-like and spacer sequences but not regionII (nucleotides 101 to 236) directs Nrd1-dependent termination.Indeed, this smaller region (SNR65short) causes moderate coppersensitivity that is relieved by the nrd1-5 mutation (Fig. 1C).

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FIG. 2. Termination-polyadenylation elements of snR13 and snR65 pre-snoRNAs. Nucleotide positions relative to the transcription start site and mature 5' end are shown above and below the sequences for snR13 and snR65, respectively. Lowercase letters indicate nucleotides contained within mature snR13, while uppercase letters indicate nucleotides downstream of the mature 3' end of either RNA. Also shown is a portion of the NRD1 5' UTR (nucleotides –229 to –173 relative to the start codon) that contains a Nrd1-responsive element (40). Sequences that are identical in two or more of the genes are in boldface. Residues at which mutations decrease terminator function in the context of the ACT/CUP fusion gene intron, either singly or in combination (see Table 1), are boxed. SNR13 and SNR65 polyadenylation sites in the context of the ACT/CUP fusion gene 3' UTR are indicated by filled stars (major sites) or open stars (additional sites).

Northern analysis of RNA from ACT/CUP reporter genes with SNR65terminator sequences in the intron (Fig. 1D) corroborates theresults of the copper growth assay. The SNR65short insertioncauses four- to fivefold reduced accumulation of full-lengthACT/CUP mRNA relative to endogenous actin mRNA in a wild-typestrain, while in the nrd1-5 strain the insertion causes onlyan approximately twofold reduction (compare lanes 1, 4, and5). The SNR65long insertion results in complete loss of full-lengthmRNA and the accumulation, instead, of a truncated transcript,and this pattern does not change in a nrd1-5 mutant strain (comparelanes 1, 3, and 6). We conclude that the SNR65 terminator consistsof proximal elements that are relatively Nrd1 dependent (regionI) and distal elements that are relatively Nrd1 independent(region II).

To identify nucleotides critical for SNR65 terminator function,we generated a pool of mutated DNA fragments containing theentire SNR65/RPS14A intergenic region by error-prone PCR andintroduced them into the ACT/CUP fusion gene intron in the forwardorientation by in vivo homologous recombination. Yeast transformantsthat survived on plates containing 0.15 mM copper, a concentrationrestrictive for the wild-type insert, were selected. The ACT/CUPplasmid was rescued from 17 copper-resistant transformants,plasmid linkage of copper resistance was confirmed by retransformation,and the insert from each plasmid was sequenced. The resultsare displayed in Table 1 and Fig. 2. Interestingly, 14 of the17 plasmids have mutations affecting the UA dinucleotide atpositions 260 and 261, and all 17 have at least one mutationin region II. Mutation of A251, U260, or A261 alone is sufficientto allow growth on 0.15 mM copper. Growth on 0.7 mM copper wasobserved only when additional mutations are present, eitheranother mutation in region II (A283G) or several mutations inregion I (U166A, C172A) and the spacer (U198 ${Delta}$ ). The failure toobtain mutations in region I alone suggests that region II isthe major determinant for termination on SNR65 and/or that regionI of SNR65 has redundant elements not easily disrupted by randommutagenesis.

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TABLE 1. cis-acting readthrough mutations in the SNR65 terminator

Polyadenylation of mRNA directed by snoRNA terminators. To test if a snoRNA terminator can function from within an mRNA3' UTR, the SNR13 and SNR65 terminators were placed individually,in both orientations, between the stop codon and polyadenylationsite of the ACT/CUP fusion gene in place of deleted CUP1 3'UTR sequences (Fig. 3A). In no case did the insertion conferstrong copper sensitivity on the cup1 ${Delta}$ strain, indicating thatfunctional ACT/CUP mRNA production was not significantly impaired.However, the SNR13 forward insert did result in slower growthon 0.7 to 1.0 mM copper plates, which could be rescued by amutation in the gene for Nrd1 (data not shown). Total cellularRNA from the transformants was analyzed by Northern blottingwith a probe complementary to CUP1 (Fig. 3B). In the reverseorientation, the SNR13 insert increases the length of the ACT/CUPmRNA but apparently does not result in any new 3' ends (Fig.3B, compare lanes 1 and 3). This result was expected since thereverse orientation should encode no 3'-end formation elements.There does appear to be a decrease in the steady-state levelof the mRNA, perhaps because the altered 3' UTR sequence destabilizesthe transcript.

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FIG. 3. (A) Scheme for mapping of functional polyadenylation sites in snoRNA terminators. The CUP1 3' UTR (157 nucleotides) was deleted prior to insertion of either the SNR13 or the SNR65 terminator at the site of the deletion. (B) Northern analysis of transcripts from ACT/CUP fusion gene alleles. Total cellular RNA was extracted from the 46 ${alpha}$ cup1 ${Delta}$ strain carrying plasmid-borne ACT/CUP alleles with the following modifications: lanes 1 and 6, deletion of 157 nucleotides of the CUP1 3' UTR ( ${Delta}$ ); lanes 2 and 3, insertion of SNR13 nucleotides +125 to +232 in the forward (F; lane 2) and reverse (R; lane 3) orientations; lanes 4 and 5, insertion of SNR65 nucleotides +101 to +317 in the reverse (lane 4) and forward (lane 5) orientations. The arrow and dashed line mark the position of mRNA with only the CUP1 3' UTR deletion. The blot was probed with ³²P-labeled oligonucleotide 3'CUP-2, complementary to the CUP1 coding region. Ethidium bromide staining of the portion of the filter containing the large and small rRNAs is shown below the lane numbers, for normalization.

In the forward orientation, the SNR13 insert results in theformation of two sets of transcripts in roughly equal amounts,one of the size expected for use of the PGK1 polyadenylationsite present in the ACT/CUP fusion gene and a shorter set ofthe size expected if the snoRNA terminator was utilized (Fig.3B, lane 2). To determine if the shorter transcripts are polyadenylated,and to map the sites of polyadenylation, cDNA was prepared fromthe RNA with reverse transcriptase and an oligo(dT) primer.The resulting cDNA was amplified by PCR with the oligo(dT) primerand a CUP1-specific reverse primer, cloned, and sequenced. Strikingly,the polyadenylation sites map to the downstream end of regionII of the SNR13 insert (Fig. 2). Of 19 clones sequenced, 11had +211 and two had +210 as the last nucleotide before thepoly(A) tail. The remaining six clones correspond to six differentpoly(A) sites from position +218 to position +277. All but twoof the sites are within SNR13 sequences. Curiously, none ofthe clones correspond to mRNAs cleaved and polyadenylated atthe PGK1 poly(A) site, even though such transcripts appear tobe similar in abundance to transcripts ending in SNR13 sequences(Fig. 3B, lane 2). The RT-PCR also lacked products of the lengthexpected for the PGK1 poly(A) site, which suggests that theRT-PCR conditions we used favored synthesis of the shorter cDNAs.Our results indicate that the SNR13 terminator can direct polyadenylationwhen present in the 3' UTR of an mRNA-encoding gene.

The SNR65 terminator also elicited polyadenylation in the ACT/CUP3' UTR. The 217-bp fragment bearing the SNR65 terminator containsthe RPS14A cleavage-and-polyadenylation site in the oppositeorientation, so it was expected to direct 3'-end formation ineither orientation. Indeed, both the forward and reverse orientationsof the insert direct the formation of shorter transcripts thanare seen in the absence of an insert (Fig. 3B, compare lanes4 and 5 with lane 6), indicative of cleavage and/or terminationwithin the insert. Both the SNR65 and RPS14A terminator elementsappear to be more efficient than the SNR13 terminator in thecontext of the ACT/CUP 3' UTR, as little or no utilization ofthe PGK1 cleavage-and-polyadenylation site is apparent. Thefact that the transcripts produced from the reverse orientationof the SNR65 insert are approximately the same length as thoseproduced from the forward orientation of the SNR13 insert (Fig.3B, compare lanes 2 and 4) indicates that the RPS14A polyadenylationsite is about 100 nucleotides downstream of the stop codon,between regions I and II of the SNR65 terminator (Fig. 1A).Indeed, of seven cDNAs analyzed for the reverse SNR65 insert,six have poly(A) sites between +205 and +220 (Fig. 2, in theopposite strand of that shown). The seventh clone has a poly(A)site at position +181. Thus, the SNR65 and RPS14A terminatorsare indeed interdigitated on opposite strands.

The polyadenylation sites for the SNR65 terminator were mappedby the same method as that described for the SNR13 terminator,and the results are displayed in Fig. 2. Eight cDNA clones wereanalyzed; four had poly(A) sites at position +303, and the remainderhad sites between +287 and +297. Thus, as for SNR13, the SNR65poly(A) sites are at the downstream end of region II. It thereforeappears that at least two snoRNA terminators contain cleavageand polyadenylation signals at their downstream ends, althoughwe cannot exclude the possibility that the poly(A) tail derivesfrom termination followed by cleavage-independent polyadenylationby Trf4 (see Discussion).

trans-acting terminator readthrough mutations in Pol II subunits. We have also used the ACT-CUP reporter to select for mutationsin trans-acting factors that induce readthrough of the SNR13terminator (38-40). The factors thus identified include twoRNA-binding proteins, Nrd1 and Nab3, a helicase, Sen1, and aprotein phosphatase, Ssu72. Nrd1 and Sen1 bind to the C-terminaldomain of the largest subunit of Pol II, Rpb1 (42, 48), whileSsu72 binds to the second largest Pol II subunit, Rpb2 (16),and utilizes the Pol II C-terminal domain as a substrate (29).Nab3 binds to Nrd1 (10). A plausible model for the functionof these four factors is that Nrd1 and Nab3 recognize terminatorsequences in the nascent transcript, possibly assisted by Sen1,and transmit the termination signal to Pol II, perhaps via Ssu72.The mechanism by which Pol II receives the termination signalis unknown, and no mutations in Pol II subunits had been obtainedin the readthrough selection.

Here we report the identification of terminator readthroughmutations in two subunits of Pol II. Selection for spontaneouscopper-resistant mutants with a haploid cup1 ${Delta}$ yeast strain bearingthe ACT/CUP fusion gene with the SNR13 terminator inserted inthe intron yielded a cold-sensitive mutation that was complementedby a plasmid bearing RPB11, the gene encoding the second smallestsubunit of Pol II (46). The RPB11 locus was amplified from thecold-sensitive strain by PCR and sequenced, revealing a missensemutation that substitutes glycine for glutamate at residue 108of the 120-amino-acid Rpb11 protein (rpb11-E108G). When clonedinto a low-copy-number plasmid and transformed into a wild-typeRPB11 strain, the rpb11-E108G allele dominantly confers readthroughof the SNR13 terminator (data not shown), as well as the poly(A)site/terminator from the protein-coding CYC1 gene (Fig. 4A).These results confirm that the rpb11-E108G mutation is solelyresponsible for the copper-resistant phenotype and demonstratethat the substitution in Rpb11 affects recognition of both asnoRNA and an mRNA terminator.

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FIG. 4. Terminator readthrough mutations in the Rpb11 and Rpb3 subunits of Pol II. (A) Scheme for selecting trans-acting mutations that induce readthrough of either the snR13 snoRNA gene terminator or the Cyc1 protein-coding gene terminator. In the examples shown, the rpb11-E108G mutation induces readthrough of the SNR13 terminator when present in the genome and dominantly induces readthrough of the CYC1 terminator when present on a low-copy-number plasmid. (B) Readthrough substitutions isolated in Rpb11 are located at the C terminus and are shown above an alignment of Rpb11 orthologs. Only single-residue substitutions are shown (see Table 2). Numbers refer only to residues in the S. cerevisiae protein. Conserved hydrophobic residues in the ${alpha}$ -helical segment are boxed. Humans have several isoforms of Rpb11 that differ at the C terminus (21); isoform a is shown. (C) The readthrough substitution isolated in Rpb3 is located at the N terminus and is shown above an alignment of Rpb3 orthologs. The most-conserved residues are boxed.

The Rpb11-E108G substitution lies near the C-terminal end ofa seven-turn ${alpha}$ -helix that forms a coiled-coil interaction withthe third largest subunit of Pol II, Rpb3 (12). To search forother substitutions in the Rpb3/11 heterodimer that interferewith termination, RPB3 and RPB11 were randomly mutated by error-pronePCR and introduced into the reporter strain by transformation.Transformants were selected for dominant induction of readthroughof either the SNR13 or the CYC1 terminator. As a control, randomlymutated RPB7 was put through the same selection. The Rpb7 subunitmaps to a different face of Pol II than the Rpb3/11 heterodimer(2, 5) (Fig. 5). Very few copper-resistant colonies were obtainedafter transformation with mutated RPB7, and these strains retainedcopper resistance after selection against the URA3-marked RPB7plasmid, indicating that readthrough was due to spontaneousmutations in the genome or ACT/CUP reporter. In contrast, hundredsof copper-resistant colonies were obtained after transformationwith mutated RPB11. When 15 of these candidate RPB11 mutantstrains were subjected to selection against the URA3-markedplasmid, all 15 lost copper resistance, indicating that RPB11plasmid-linked mutations could readily be selected. Copper-resistantstrains were also readily obtained with mutated RPB3. The RPB3mutant candidates were not subjected to the plasmid linkagetest, but sequencing of the plasmid-borne RPB3 gene from 12candidates revealed that they all contain the same substitution(see below).

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FIG. 5. Shown are top (A) and rear (B) orthogonal views of 12-subunit yeast Pol II with a transcription bubble mimic in the active site (27). DNA (pale yellow) enters from the top in panel A and exits toward the viewer. RNA (orange) exits through a pore that is covered in part by the lid (magenta). Rpb3 (copper) and Rpb11 (gold) are at the back of Pol II, while Rpb7 (pale green) extends from the side. Rpb11 residue E108 is red, while L111 and L114 are gold. (Residues 115 to 120 are not resolved in the crystal structure.) Rpb3 residue K9 is bright green. Rpb11-E108 is close to Rpb3-K9 in the back view. The C termini of Rpb3 and Rpb11 are shown as ribbons to emphasize the coiled-coil structure. This figure was created with PyMOL (DeLano Scientific) by using pdb file 1Y1W (www.rcsb.org/pdb).

Sequencing of 29 RPB11 and 12 RPB3 readthrough alleles identifiedthe substitutions listed in Table 2. By far the most commonmutation selected in RPB11 is the E108G substitution that wasalso obtained in the genome-wide spontaneous mutation selection,suggesting that this substitution provides an optimal balancebetween readthrough of the intronic terminator and recognitionof native terminators. Two other substitutions were recoveredat position 108, E108K and E108V. The rarity of these substitutionsrelative to E108G could be due to a semidominant detrimentaleffect on cell growth. Consistent with this notion, a test ofgrowth phenotypes in the absence of wild-type RPB11 revealedthat an rpb11-E108G mutant strain grows normally at 30°C,but the rpb11-E108K mutant strain is not viable (Table 2), despitethe dominant effect of the rpb11-E108K allele on terminatorrecognition. The difference in function of Rpb11-E108G and Rpb11-E108Ksuggests that the substitution of glycine for glutamate simplyresults in loss of a favorable contact, whereas substitutionof lysine for glutamate results in a steric and/or ionic clashin addition to the loss of a specific contact.

Strikingly, all other mutations selected in RPB11 result inchanges C terminal to E108 (Fig. 4B). The most common of thesewere the substitutions L111P and L114P. In addition, nonsensemutations were obtained at codons 109, 112, and 114, eitheralone or in combination with a substitution (Table 2). Thus,alterations in the structure of the extreme C-terminal segmentof Rpb11 appear to result in inefficient recognition of terminatorsby Pol II. The fact that the readthrough mutations are dominantindicates that the alterations in Rpb11 do not prevent assemblyinto Pol II, nor do they strongly inhibit initiation or elongationby Pol II.

All 12 Rpb3 mutants identified in the selection encode a substitutionof glutamate for lysine 9 (K9E, Fig. 4C). Remarkably, K9 ofRpb3 lies within a few angstroms of Rpb11-E108 in crystal structuresof yeast Pol II (Fig. 5). Rpb3-K9 is not part of the C-terminal ${alpha}$ -helix that forms a coiled coil with Rpb11; rather, it is ina ß-sheet that cradles the C terminus of the Rpb11 ${alpha}$ -helix. The asymmetry of the selected substitutions in Rpb11and Rpb3 suggests that it is not simply disruption of the coiledcoil that results in readthrough, but rather alterations inthe surface at one end of the coiled coil. A different domainof Rpb3 was previously implicated in the activation of transcription(41); our mutations are the first evidence for a function ofthe Rpb3/11 heterodimer in termination.

The basic side chain of Rpb3-K9 and the acidic side chain ofRpb11-E108 have the potential of forming an ionic interaction.However, this potential is not conserved in several of the knownorthologs of yeast Rpb3/11 (Fig. 4). Furthermore, the recessivelethal growth defect of an rpb11-E108K mutant strain is notrescued by transformation with rpb3-K9E on a low-copy-numberplasmid (data not shown), suggesting that an ionic interactionbetween these two residues is not sufficient for normal Rpb3/11activity.

DISCUSSION

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Discussion

While it has been known for some time that termination of transcriptionby Pol II on protein-coding genes is coupled to cleavage andpolyadenylation, the elements that direct termination of PolII on noncoding genes are poorly characterized. Furthermore,the mechanism by which the termination signal of any gene istransduced from factors that recognize the terminator elementsto Pol II is obscure. This study illuminates both the natureof cis-acting elements that induce termination on yeast snoRNAgenes and the way in which Pol II receives the signal that suchelements are present.

The SNR13 and SNR65 terminators contain polyadenylation signals. The terminators from two yeast snoRNA genes, SNR13 (7, 33, 39)and SNR47 (7), have previously been studied in some detail.Both terminators are bipartite in structure, with functionalelements centered roughly 45 bp apart. We have now characterizeda third snoRNA terminator, that of SNR65. Interestingly, ittoo is bipartite, but the region I and region II elements arecentered about 95 bp apart, due to the presence of a 50-bp spacersequence. Within this spacer lies the cleavage-and-polyadenylationsite for the convergently transcribed gene RPS14A. Thus, separationof regions I and II may be an adaptation to allow interdigitatedterminators on opposite strands. Such flexibility in terminatorarchitecture contributes to the difficulty in identifying terminatorsby sequence alone. Interestingly, regions I of the SNR13 andSNR65 terminators, and the NRD1 autoregulatory element, sharefairly extensive sequence similarity (Fig. 2), which suggeststhat natural terminators are more than just a collection oflow-affinity Nrd1 (GUAA) and Nab3 (UCUU) binding sites (7).

We previously noted that region II of the SNR13 terminator hassequence similarity to yeast cleavage-and-polyadenylation sites(39). Here we show that both the SNR13 and SNR65 terminatorsdirect polyadenylation when placed in the 3' UTR of a reportergene. In either case, polyadenylation occurs at the downstreamboundary of region II, within a few nucleotides of sites predictedby the algorithm of Graber et al. (20), which was developedto identify cleavage-and-polyadenylation sites in protein-codinggenes. We therefore presume that these polyadenylated 3' endsare produced by the cleavage and polyadenylation factors thatact on mRNAs. While we cannot rule out the possibility thatthese 3' ends are generated by termination and are subsequentlypolyadenylated by Trf4, which marks nuclear RNAs for exosomaldegradation (25, 30, 43, 47), we consider this scenario unlikelysince the transcripts accumulate to high levels and are translatedefficiently, judging by their ability to confer copper resistance.Furthermore, our observations are consistent with the fact thatseveral cleavage and polyadenylation factors have been implicatedin termination on snoRNA genes. These include CFIA subunitsRna14 and Rna15 (17, 33) and holo-CPF subunits Pti1, Ref2, Ssu72,and Swd2 (9, 14-16, 19, 34, 39).

Earlier studies detected polyadenylated forms of two noncodingRNAs, telomerase RNA (8) and U2 snRNA (1). Approximately 5 to10% of telomerase RNA is polyadenylated in wild-type yeast cells,whereas polyadenylation of U2 RNA is observed only when processingof precursor U2 RNA by Rnt1 endonuclease is inhibited. SeveralsnoRNA genes produce extended and polyadenylated transcriptswhen the nuclear exosome is inhibited by deletion of the genefor the Rrp6 subunit, and this polyadenylation is dependenton Pap1, the poly(A) polymerase that acts on mRNAs (44). Itis not clear if the polyadenylation sites of these noncodingRNAs map to the 3' ends of their terminator sequences and thusare mechanistically similar to cleavage and polyadenylationdirected by the SNR13 and SNR65 terminators.

It is intriguing that the SNR65-RPS14A intergenic region didnot confer copper sensitivity when inserted into the ACT/CUPfusion gene intron in the RPS14A orientation, yet it efficientlydirected cleavage and polyadenylation when inserted into theACT/CUP 3' UTR. One interpretation of this result is that theRPS14A cleavage-and -polyadenylation signal can only be recognizedafter Pol II has transcribed several hundred nucleotides ofRNA. In contrast, the CYC1 cleavage-and-polyadenylation signalstrongly decreases ACT/CUP gene expression when inserted intothe intron (Fig. 4A). The CYC1 coding region is only 330 bplong, while the RPS14A coding region (with intron) is 721 bplong. Thus, the dichotomous behavior of the terminator elementsof these two genes may reflect their adaptation to differentgene lengths. This model is consistent with the finding thatNrd1, Nab3 (34), and Sen1 (E.J.S. and D.A.B., unpublished data)exhibit high occupancy over both the 5' and 3' ends of testedprotein-coding regions, suggestive of recruitment upon initiationof transcription, while cleavage and polyadenylation factorspeak primarily over the 3' end of coding regions, implying recruitmentonly after significant elongation (28, 34). We propose thatthe Nrd1-dependent termination pathway is most active withina few hundred base pairs of the transcription start site, whilethe cleavage-and-polyadenylation-dependent termination pathwayoperates primarily at sites several hundred base pairs downstreamof the start site. Intermediate sites, such as the CYC1 poly(A)site, might utilize components of both pathways.

Substitutions in the Rpb3/11 heterodimer cause terminator readthrough. How might the selected substitutions in the Rpb3/11 heterodimerinduce terminator readthrough? The location of the substitutionson the rear surface of Pol II suggests that they may interferewith the interaction of Pol II with an extrinsic terminationfactor. Termination of Pol II transcription appears to requirethe binding of protein factors to terminator elements in thenascent transcript (3, 37). Although the Rpb3/11 mutations aredistant from the RNA exit pore (Fig. 5), a basic channel thatmay provide a path for the nascent transcript ("groove 2" ofreference 11) leads from the RNA exit pore to the vicinity ofRpb3/11. Therefore, a termination factor that binds to the nascenttranscript may contact Pol II at the Rpb3/11 heterodimer. Sucha factor may induce termination by causing an allosteric changein the elongating polymerase, or it may reach around the backof the enzyme and interact more directly with macromoleculesin or near the active site. It is interesting that the Rpb3/11heterodimer plays a central role in the interaction of Pol IIwith the 21-subunit Mediator protein complex (13). While Mediatorfunctions in initiation of transcription and its involvementin termination has not been proposed, one can imagine that bindingof Mediator to Pol II at the end of a gene may serve to coupletermination to reinitiation.

There are several other possible candidates for a factor thatinteracts with Rpb3/11 to promote termination. Because our mutationscause readthrough of both a snoRNA and an mRNA terminator, factorsknown to be common to both pathways are of particular interest.Such factors currently include the cleavage and polyadenylationfactors mentioned above, as well as the helicase Sen1 (38, 40;E.J.S. and D.A.B., unpublished). Given that the Rpb3/11 heterodimeris homologous to the N-terminal domains of the ${alpha}$ subunit homodimerin bacterial RNA polymerase, it is intriguing that mutationsin the Escherichia coli ${alpha}$ subunit C-terminal domain have beenshown to cause readthrough of terminators that are dependenton the helicase rho (26). These observations raise the possibilitythat the signaling pathway for transcription termination ineukaryotes and prokaryotes may employ analogous interactionsbetween a transcript-associated helicase and the upstream endof RNA polymerase.

ACKNOWLEDGMENTS

We thank E. Settles for technical assistance; J. Keck, M. Lopper,M. Killoran, and S. Butcher for help in creating Fig. 5; andR. Gourse, W. Ross, R. Landick, J. Dahlberg, and J. S. Butlerfor discussions and comments on the manuscript.

This work was supported by Public Health Service grant GM44665.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biomolecular Chemistry, University of Wisconsin Medical School, 1300 University Avenue, Madison, WI 53706-1532. Phone: (608) 262-1475. Fax: (608) 262-5253. E-mail: dabrow{at}wisc.edu.

REFERENCES

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