Stimulation of RNA polymerase II transcript cleavage activity contributes to maintain transcriptional fidelity in yeast

© 2007 The Authors Genes to Cells (2007) 12, 547–559Journal compilation © 2007 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

547DOI: 10.1111/j.1365-2443.2007.01072.x

Blackwell Publishing IncMalden, USAGTCGenes to Cells1356-9597© Blackwell Publishing LtdXXX Original ArticlesRole of transcript cleavage in fidelityH Koyama et al.Stimulation of RNA polymerase II transcript cleavage activity contributes to maintain transcriptional fidelity in yeast

Hiroshi Koyama1, Takahiro Ito1, Toshiyuki Nakanishi2 and Kazuhisa Sekimizu1,*1Department of Microbiology, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan2New Product Research Laboratories III, Tokyo R&D Center, Daiichi Pharmaceutical Co., Ltd. Japan

The transcription elongation factor S-II, also designated TFIIS, stimulates the nascent transcriptcleavage activity intrinsic to RNA polymerase II. Rpb9, a small subunit of RNA polymerase II,enhances the cleavage stimulation activity of S-II. Here, we investigated the role of nascent tran-script cleavage stimulation activity on the maintenance of transcriptional fidelity in yeast. In yeast,S-II is encoded by the DST1 gene. Disruption of the DST1 gene decreased transcriptional fidelityin cells. Mutations in the DST1 gene that reduce the S-II cleavage stimulation activity led todecreased transcriptional fidelity in cells. A disruption mutant of the RPB9 gene also had decreasedtranscriptional fidelity. Expression of mutant Rpb9 proteins that are unable to enhance the S-IIcleavage stimulation activity failed to restore the phenotype. These results suggest that both S-IIand Rpb9 maintain transcriptional fidelity by stimulating the cleavage activity intrinsic to RNApolymerase II. Also, a DST1 and RPB9 double mutant had more severe transcriptional fidelitydefect compared with the DST1 gene deletion mutant, suggesting that Rpb9 maintains transcriptionalfidelity via two mechanisms, enhancement of S-II dependent cleavage stimulation and S-IIindependent function(s).

IntroductionLoss of transcriptional fidelity during mRNA synthesiscauses the production of mutated proteins. Thus, excessivetranscriptional errors impair cellular function. Taddei et al.(1997) proposed that selective elimination of oxidizednucleoside triphosphates from the cellular nucleotidepool is important for maintaining transcriptional fidelityin Escherichia coli cells. During DNA synthesis, DNApolymerases exert 3′→ 5′ exonuclease activity to proof-read the newly synthesized DNA (Joyce & Steitz 1994).By analogy, several studies have proposed that the 3′→ 5′nuclease activity intrinsic to RNA polymerases is involvedin transcriptional proofreading (Erie et al. 1993; Jeon &Agarwal 1996; Thomas et al. 1998; Lange & Hausner2004). Both prokaryotic and eukaryotic RNA poly-merases possess the 3′→ 5′ nuclease activity (Wang &Hawley 1993; Orlova et al. 1995). In E. coli, transcriptionelongation factors GreA and GreB activate the nucleaseactivity, leading to increased transcriptional fidelityin vitro (Erie et al. 1993). In eukaryotes, the 3′→ 5′ nuclease

activity of RNA polymerase II is stimulated by tran-scription elongation factor S-II (Izban & Luse 1992;Reines 1992). This process is further stimulated byRpb9, a small subunit of RNA polymerase II (Awreyet al. 1997). There is no genetic evidence, however, tosupport the notion that stimulation of the 3′→ 5′nuclease activity intrinsic to the RNA polymerasescontributes to maintain transcriptional fidelity in eitherprokaryotic or eukaryotic cells. Moreover, the physiologicrelevance of the mechanisms that underlie the mainte-nance of transcriptional fidelity, including the eliminationof oxidized nucleotides, the 3′→5′ nuclease activity ofRNA polymerases, and the stimulation of this nucleaseactivity, remain uncertain.

Transcription elongation factor S-II/TFIIS wasoriginally purified as a stimulatory protein of RNApolymerase II from mouse Ehrlich ascites tumor cells(Natori et al. 1973; Sekimizu et al. 1979). During RNAsynthesis, RNA polymerase II is arrested by varioustranscriptional blocks, and in some cases, backtracks byseveral nucleotides along the template DNA. As a result,the 3′-end of the transcript dissociates from the catalyticcenter of RNA polymerase II (Fish & Kane 2002). Extensivebiochemical studies revealed that S-II reactivates RNA

Communicated by : Hiroshi Handa*Correspondence: E-mail: [email protected]

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Genes to Cells (2007) 12, 547–559 © 2007 The AuthorsJournal compilation © 2007 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

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polymerase II to read-through the arrest sites. WhenRNA polymerase II is arrested, the 3′→ 5′ nucleaseactivity intrinsic to RNA polymerase II is stimulated byS-II to re-align the 3′-end of the transcript with thecatalytic center, and then RNA polymerase II resumestranscription. Thus, stimulation of the 3′→ 5′ nucleaseactivity by S-II is required for the S-II mediated read-through. This 3′→ 5′ nuclease activity intrinsic to RNApolymerase II is designated “cleavage” activity. The cleav-age stimulation activity of S-II is not sufficient, however,for S-II mediated read-through; Cipres-Palacin & Kane(1994) and Awrey et al. (1998) demonstrated that severalmutant S-II proteins possess the cleavage stimulationactivity, but do not promote read-through in vitro. There-fore, S-II is necessary not only for cleavage stimulation,but also for the transcriptional resumption step after thecleavage reaction in vitro. In other words, the read-throughstimulation activity of S-II consists of two functions,stimulation of the cleavage reaction and transcriptionalresumption after the cleavage reaction. Although themechanism for the reactivation of RNA polymerase IIafter the cleavage reaction has not been characterized,several reports propose that a conformational change inRNA polymerase II might be necessary and that S-II isrequired for the change (Awrey et al. 1998).

In Saccharomyces cerevisiae, S-II is necessary for inductionof the SSM1/SDT1 and the IMD2/PUR5 genes (Shaw& Reines 2000; Shimoaraiso et al. 2000). The SSM1gene harbors transcriptional arrest sites. The IMD2 genemight contain the arrest sites, and mutations in thetranscription elongation machinery render yeast cellsdefective in IMD2 gene induction. The cleavage, but notthe read-through stimulation, of S-II is necessary forIMD2 gene induction in yeast (Ubukata et al. 2002). Thus,S-II mediated transcriptional resumption after cleavageis not essential for IMD2 gene induction. These resultssuggest that when RNA polymerase II reads through tran-scriptional arrest sites in yeast, a factor(s) other than S-IImight be involved in the transcriptional resumption afterthe S-II mediated cleavage reaction (Ubukata et al. 2002).

Besides transcription arrest at specific sites on templateDNA, RNA polymerase II also stops transcription tem-porarily when incorrect ribonucleotides are incorpo-rated into the nascent transcripts (Thomas et al. 1998). Invitro studies demonstrated that S-II enhances the excisionof these mis-incorporated nucleotides by stimulating thecleavage activity of RNA polymerase II ( Jeon & Agarwal1996). These results suggest that S-II contributes totranscriptional fidelity via transcriptional proofreadingmediated by cleavage stimulation. Previously, we reportedthat transcriptional fidelity is reduced in DST1 gene-disrupted yeast (Koyama et al. 2003). There is no genetic

evidence, however, that S-II stimulation of RNA poly-merase II cleavage activity is essential for the cellularfunction of S-II in transcriptional fidelity.

Rpb9, a small subunit of RNA polymerase II, func-tionally interacts with S-II (Hemming et al. 2000;Hemming & Edwards 2000). Rpb9 promotes thecleavage reaction by enhancing the response of RNApolymerase II to S-II. In other words, Rpb9 enhancesthe cleavage stimulation activity of S-II. Moreover, inthe absence of Rpb9, S-II is unable to exert its read-through stimulation activity (Awrey et al. 1997). TheRPB9 gene is dispensable for cell growth under usualculture conditions in S. cerevisiae, but loss of the RPB9gene causes sensitivity to nucleotide-depleting drugs(6-azauracil and mycophenolic acid), which is similarlyinduced by DST1 gene disruption (Woychik et al. 1991;Exinger & Lacroute 1992; Nakanishi et al. 1992; VanMullem et al. 2002). Recently, Rpb9 was shown to beinvolved in the maintenance of transcriptional fidelity inyeast (Nesser et al. 2006). It remains unclear whether thecleavage stimulation activity of Rpb9, which depends onS-II, serves to maintain transcriptional fidelity.

Here, we investigated whether the cleavage stimulationactivity of S-II and Rpb9 contributes to transcriptionalfidelity by using yeast strains bearing mutations on theDST1 and/or the RPB9 genes. Mutant strains that aredefective in the maintenance of transcriptional fidelityhave increased sensitivity to oxidative stress. We discussthe possibility that the mechanism underlying the main-tenance of transcriptional fidelity conferred by stimulat-ing RNA polymerase II cleavage activity is involved inthe resistance to oxidative stress in yeast.

ResultsS-II cleavage stimulation activity is responsible for maintaining transcriptional fidelity in yeast

We previously reported that DST1 disrupted mutantyeast had reduced transcriptional fidelity and that theRNA polymerase II binding region in S-II is critical forthe maintenance of transcriptional fidelity (Koyama et al.2003). During transcription elongation in vitro, S-IIstimulates the nascent RNA cleavage activity intrinsicto RNA polymerase II (Izban & Luse 1992). S-II alsostimulates RNA polymerase II to read-through tran-scriptional arrest sites (Reines et al. 1989). This processcomprises two distinct activities of S-II: stimulation ofRNA cleavage and of transcriptional resumption afterthe cleavage reaction (Awrey et al. 1998). In this report,we call the latter activity “transcriptional resumptionstimulation activity.” To determine which of the two

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S-II activities is responsible for maintaining transcriptionalfidelity in yeast, we examined transcriptional fidelityin yeast strains expressing a panel of point-mutated S-IIproteins that have reduced cleavage and/or read-throughstimulation activity in vitro (Awrey et al. 1998; Ubukataet al. 2002). Awrey et al. used truncated form of S-IIproteins bearing point mutation(s) to evaluate the in vitroactivities. The truncated S-II proteins contain the C-terminal half of the S-II protein (131–309 amino acidresidues). In the present study, we used full-length S-IIproteins bearing point mutation(s) which would havesimilar transcription activities to the truncated proteinsbearing the same mutation(s), since the C-terminal half

of S-II is sufficient to promote both cleavage andread-through with efficiencies nearly identical to thefull-length S-II (Cipres-Palacin & Kane 1994; Nakanishiet al. 1995; Awrey et al. 1998). The activity scores of theseS-II mutant proteins were defined according to Awreyet al. (Fig. 1A).

To evaluate transcriptional fidelity in these S-II mutantstrains, we used a genetic reporter assay with a mutatedlacZ gene in which codon 461 (GAG), encoding gluta-mate in the wild-type lacZ protein, was replaced with astop codon (TAG), as described in our previous report(Koyama et al. 2003). Active β-galactosidase will not beexpressed if the mutated lacZ gene (pMLac) is correctly

Figure 1 The cleavage stimulation activity of S-II is responsible for both transcriptional fidelity maintenance and oxidative stressresistance in yeast. (A) Transcriptional error rates in HKY01 (dst1∆), YPH499 (DST1 wild-type), or cells expressing point-mutated S-IIproteins (Mt1-7, 9). The error rate of HKY01 was defined as 100%. Three independent experiments were performed in duplicate, andmean values with standard errors of the mean are shown. The score of the cleavage and the read-through stimulation activities of thesepoint-mutanted S-II proteins were defined previously (Awrey et al. 1998). (B) Serial dilutions of the DST1 mutant strains were spottedon to plates containing either 1.2 mm hydrogen peroxide or 25 µm menadione (indicated as “+”), and incubated at 30 °C. Plate imageswithout these drugs are also shown (indicated as “–”).

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transcribed. In contrast, active β-galactosidase will beexpressed if RNA polymerase II mis-incorporates anincorrect nucleotide at the mutated site in the lacZ gene.For example, if GTP is mis-incorporated instead ofUTP at the mutated TAG stop codon, the resulting codonGAG will be translated as a glutamate correspondingto the wild-type lacZ protein. Thus, in this assay, reducedtranscription fidelity in yeast cells results in increasedβ-galactosidase activity. We also used the wild-type lacZgene (pWLac) as a reporter in place of the mutated gene(pMLac) to determine the lacZ gene expression levelin the mutant yeast strains. The transcriptional error ratewas defined as ratio of the β-galactosidase activity fromthe mutated lacZ gene to that from the wild-type lacZ gene.In this assay, to compensate for the difference in the lacZgene expression level between strains used in this study,we evaluated transcriptional error rate after normalizationby the lacZ gene expression level calculated by usingthe wild-type lacZ gene as a reporter. As previouslydescribed, the error rate in the dst1∆ cells (HKY01) wastenfold higher than that in the DST1 wild-type strain(YPH499). The error rate in Mt1, 4, 5, 6, 7, or 9-expressingcells was almost the same as that in YPH499, whereas Mt2or Mt3-expressing cells had a much higher error ratethan YPH499. Mt5 protein had reduced read-throughstimulation activity, but did have strong cleavage stimulationactivity. Therefore, the transcriptional resumption stimu-lation activity of S-II was compromised in this mutantprotein. The error rate in the Mt5-expressing cells was aslow as that in YPH499, suggesting that the S-II tran-scriptional resumption stimulation activity is not requiredfor maintaining transcriptional fidelity. In contrast, Mt3protein expression, which was defective in cleavage stimu-lation activity in vitro, failed to restore the high error ratein the DST1 null mutant strain. These results suggest thatthe S-II cleavage activity, but not the S-II transcriptionalresumption stimulation activity, is responsible for main-taining transcriptional fidelity in yeast.

We previously reported that dst1∆ cells are sensitive tooxidative stress and have reduced transcriptional fidelity,and that the region of S-II protein essential for maintain-ing transcriptional fidelity coincides with the region thatconfers resistance to oxidative stress (Koyama et al. 2003).In the present study, we tested whether cells expressingpoint mutations of S-II, leading to reduced transcriptionalfidelity, are sensitive to oxidative stress. As previouslydescribed, dst1∆ cells (HKY01) were more sensitive tothe oxidants menadione and hydrogen peroxide thanwild-type cells (YPH499) (Fig. 1B). Mt3-expressing cellswere most sensitive to the oxidants. Mt2-expressing cellswere also sensitive to the oxidants, whereas expression ofthe Mt1, 4, 5, or 9 proteins rescued the oxidant sensi-

tivity in HKY01 to a level comparable to that in YPH499.Because Mt5-expressing cells are resistant to oxidativestress in spite of a defect in the transcriptional resumptionstimulation activity, the reduction of the transcriptionalresumption stimulation activity of S-II did not inevitablycause the oxidant sensitivity. In contrast, Mt3 proteinexpression, which was defective in the cleavage stimulationactivity, failed to rescue the oxidants sensitivity. Theseresults suggest that the cleavage stimulation activity ofS-II is required for oxidative stress resistance.

The cleavage stimulation activity of Rpb9 is critical for maintaining transcriptional fidelity in yeast

Previous biochemical studies demonstrated that Rpb9,a subunit of RNA polymerase II, also stimulates thenascent RNA cleavage activity intrinsic to RNA poly-merase II, but only in the presence of S-II (Awrey et al.1997). In other words, Rpb9 enhances the S-II cleavagestimulation activity. To further understand the contribu-tion of the S-II cleavage stimulation activity to maintaintranscriptional fidelity, we examined the involvement ofRpb9 in transcriptional fidelity. The transcriptional errorrate in rpb9∆ cells was tenfold higher than that in RPB9wild-type cells (BY4742), and the defect in transcriptionalfidelity was restored by introducing a plasmid harboringthe RPB9 gene (Fig. 2A). These results indicate that Rpb9is involved in maintaining transcriptional fidelity inBY4742 yeast strains.

Recently, Nesser et al. also reported that Rpb9 func-tions to maintain transcriptional fidelity in yeast (Nesseret al. 2006). It remains unclear, however, whether S-II isinvolved in Rpb9-mediated maintenance of transcrip-tional fidelity. Previous biochemical studies indicatedthat Rpb9 is not essential for S-II to exert its cleavagestimulation activity, while Rpb9 does enhance the S-IIcleavage stimulation activity (Hemming & Edwards 2000).In contrast, Rpb9 is necessary for S-II to stimulateread-through activity. These results suggest that Rpb9is required for S-II to exert its transcriptional resumptionstimulation activity in vitro. To determine the Rpb9 acti-vities necessary for maintaining transcriptional fidelity,we investigated transcriptional fidelity in yeast cellsbearing various mutant RPB9 genes that encode mutantRpb9 proteins defective in cleavage and/or read-throughstimulation activities (Hemming & Edwards 2000). Theactivity scores of the mutant Rpb9 proteins used in thisstudy were defined according to Hemming et al. (2000).

Of the various Rpb9 mutant proteins, the 1-47 and∆65-70 proteins are defective in both cleavage andread-through stimulation activities in vitro. These mutantproteins retain the activity of Rpb9 to select the accurate



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transcriptional start site in yeast (Hull et al. 1995;Hemming & Edwards 2000). The transcriptional errorrate in rpb9∆ cells bearing plasmids encoding the 1-47mutant gene was as high as that in rpb9∆ cells bearingthe empty vector. The error rate in the ∆65-70 gene-harboring cells was also much higher than that in rpb9∆cells expressing the wild-type Rpb9. Both the R91Aand D94A proteins were defective in the transcriptionalresumption stimulation activity because these proteinslack the read-through stimulation activity, although theystill enhance the cleavage stimulation activity of S-II.Introduction of the R91A or D94A genes to the rpb9∆

cells restored transcriptional fidelity to a level comparableto that in cells expressing wild-type Rpb9. The tran-scriptional error rate in cells expressing ∆89-95, thepartial deletion mutant, which also possesses cleavagestimulation activity, but lacks read-through stimulationactivity, was halfway between that in the rpb9∆ cellsharboring the empty vector and that in the cells express-ing wild-type Rpb9. These results demonstrate that thetranscriptional resumption stimulation activity of Rpb9is not essential for maintaining transcriptional fidelity,and that the cleavage stimulation activity is responsiblefor maintaining transcriptional fidelity.

Figure 2 The S-II dependent cleavagestimulation activity of Rpb9 contributes toboth maintain transcriptional fidelity andconfer resistance to menadione in yeast.(A) Transcriptional error rates in the rpb9∆strains harboring the various mutant RPB9genes or BY4742 (RPB9 wild-type). Therpb9∆ strain harboring the empty vectorwas defined as 100%. Two independentexperiments were performed in triplicateand mean values with standard errors of themean are shown. The score of the cleavageand the read-through stimulation activitiesof these mutant Rpb9 proteins weredefined previously (Hemming & Edwards2000). (B) Serial dilutions of the rpb9∆strains harboring the mutant RPB9 geneswere spotted on to plates containing25 µm menadione (indicated as “+”), andincubated at 30 °C. A plate image withoutthe drug is also shown (indicated as “–”).(C) The survival (%) of the rpb9∆ strainsharboring the mutant RPB9 genes (1-47,∆65-70, and ∆89-95) on menadione-containing plates is represented. Meanvalues with standard errors of the meanare shown (n = 4).

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To test whether loss of the cleavage stimulationactivity of Rpb9 causes sensitivity to oxidative stress inyeast cells, we investigated colony formation of the rpb9∆cells expressing various Rpb9 mutants in the presence ofmenadione. The rpb9∆ cells were much more sensitiveto menadione than the RPB9 wild-type cells (BY4742),and the phenotype was suppressed by introducingplasmids harboring the wild-type RPB9 gene (Fig. 2B).Thus, Rpb9 confers resistance to oxidative stress in yeastcells. We next examined menadione sensitivity of cellsexpressing the mutant Rpb9 proteins defective in thecleavage stimulation activity (Fig. 2B,C). The rpb9∆ cellsharboring plasmids containing the 1-47 or ∆65-70 geneswere more sensitive to menadione than the wild-typeRPB9 cells. On the other hand, the menadione sensitivityof the rpb9∆ cells was suppressed by introducing plasmidsencoding the R91A or D94A mutant Rpb9 proteins,both of which retain cleavage stimulation activity, but areunable to enhance the read-through stimulation in vitro.Cells bearing the ∆89-95 gene were partially sensitiveto menadione compared with the wild-type RPB9 cells.The cells bearing the ∆89-95 gene were apparently moreresistant to menadione than the cells bearing the ∆65-70gene in Fig. 2B, but was more sensitive in Fig. 2C. Theobserved variance is due to experimental fluctuation.Because the R91A and D94A proteins are consideredto have defective transcriptional resumption stimulationactivity, the lack of the transcriptional resumptionstimulation activity of Rpb9 did not inevitably cause themenadione sensitivity. Furthermore, the suppression ofmenadione sensitivity by introduction of the ∆65-70or 1-47 gene was slight or not significant, suggesting thatthe lack of cleavage stimulation activity of Rpb9 resultsin menadione sensitivity in yeast.

Rpb9 contributes to transcriptional fidelity in yeast in the absence of S-II

Nesser et al. implied that Rpb9 contributes to tran-scriptional fidelity in yeast in an S-II independentmanner (Nesser et al. 2006). This, together with our presentresults, suggests that Rpb9 maintains transcriptionalfidelity via two different pathways, one that is dependenton the stimulation of cleavage by S-II and another thatis S-II independent. To test this, we compared transcrip-tional fidelity in dst1∆ rpb9∆ double mutant cells withthat in the dst1∆ cells. The transcriptional error rate inthe dst1∆ rpb9∆ cells was higher than that in the dst1∆cells (Fig. 3A), indicating that Rpb9 contributes totranscriptional fidelity in the absence of S-II.

To determine the regions of Rpb9 necessary for itsS-II independent function to maintain transcriptional

fidelity, we investigated transcriptional fidelity in dst1∆rpb9∆ cells carrying plasmids encoding a panel of mutantRPB9 genes. Cells bearing the 1-47 or ∆89-95 geneshad a higher transcriptional error rate compared withcells bearing the wild-type RPB9 gene (Fig. 3A). Incontrast, introduction of the R91A, D94A, or ∆65-70genes restored the error rate to a level comparable to thatin the wild-type RPB9 cells. These results indicate thatthe R91A, D94A, and ∆65-70 proteins maintain tran-scriptional fidelity in the absence of S-II. In other words,these three mutant proteins of Rpb9 contribute tomaintain transcriptional fidelity in an S-II independentmanner. Although the ∆65-70 protein had a slight effecton maintaining transcriptional fidelity in the presenceof S-II, this protein contributed to transcriptionalfidelity to a similar extent as the wild-type Rpb9 proteinin the background of dst1∆ (Fig. 3A). Thus, the regionsof Rpb9 necessary for transcriptional fidelity mainte-nance in the absence of S-II are distinct from the regionsrequired in the presence of S-II. In contrast, although the∆89-95 gene partially restored transcriptional fidelity inthe presence of S-II (Fig. 2A), this capability of ∆89-95was almost lost in the absence of S-II (Fig. 3A). Theseresults indicate that the C-terminal region (down toamino acid residue 47 of Rpb9) is necessary for thefunction(s) of Rpb9 to maintain transcriptional fidelityin an S-II independent manner. Furthermore, of theC-terminal region, the amino acid residues 65–70, R91,and D94 are not required, but the amino acid residues89–95 are important for maintaining transcriptionalfidelity in the absence of S-II.

Next, we tested the menadione sensitivity of dst1∆rpb9∆ cells carrying various mutant RPB9 genes. Thedst1∆ rpb9∆ cells were more sensitive to menadionethan the dst1∆ single mutant cells (Fig. 3B,C). This find-ing indicates that menadione sensitivity is caused notonly by loss of the cleavage stimulation activity of Rpb9,but also by loss of another function(s) of Rpb9 that isindependent of S-II. To determine critical regions ofRpb9 for menadione resistance in the absence of S-II,we tested whether menadione sensitivity is suppressedby introduction of the various mutant RPB9 genes.The dst1∆ rpb9∆ cells carrying the 1-47 or ∆89-95 geneswere more sensitive to menadione than dst1∆ rpb9∆ cellscarrying the wild-type RPB9 gene. Thus, in the absenceof S-II, deletion of the C-terminal regions (down toamino acid residue 47) of Rpb9 causes menadionesensitivity. The amino acid residues 89-95 have a criti-cally important role in menadione resistance. Meanwhile,when the gene encoding the R91A, D94A, or ∆65-70proteins was introduced to dst1∆ rpb9∆ cells, menadionesensitivity was restored to the level of the wild-type



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RPB9 cells. Therefore, the amino acid residues ofRpb9 from 65 to 70, R91, or D94 are not essential formenadione resistance.

Isolation of revertants for the RPB9 gene disrupted yeast

Using various mutant RPB9 genes, we found that thereis a good correlation between transcriptional fidelity andmenadione sensitivity (Figs 2 and 3). To further assess the

relationship between transcriptional fidelity and mena-dione sensitivity, we tried to isolate menadione-resistantrevertants for the dst1∆ or the rpb9∆ cells. The rpb9∆ cellswere mutagenized with ethylmethane sulfonate (EMS),and eight menadione-resistant revertant strains wereobtained from the rpb9∆ cells (Fig. 4). We could not screenthe dst1∆ cells for menadione-resistant revertant strainsbecause of technical difficulties. Among the eight revertantstrains isolated, three strains (Rev. #2, 4 and 7) showedreduced transcriptional error rate comparable with the

Figure 3 Rpb9 maintains transcriptionalfidelity and confers resistance to menadionein the absence of S-II. (A) Transcriptionalerror rates of transcription in the dst1∆rpb9∆ strains harboring the mutant RPB9genes or the dst1∆ strain. The error rateof the dst1∆ rpb9∆ strain harboring theempty vector was defined as 100%. Threeindependent experiments were performedin duplicate and mean values with standarderrors of the mean are shown. (B) Serialdilutions of the dst1∆ rpb9∆ strains harboringthe mutant RPB9 genes were spotted on toplates containing 15 µm menadione (indicatedas “+”), and incubated at 30 °C. A plateimage without the drug is also shown(indicated as “–”). (C) The survival (%) ofthe dst1∆ rpb9∆ strains harboring themutant RPB9 genes (1-47, ∆65-70, and∆89-95) on plates containing menadioneis represented. Mean values with standarderrors of the mean are shown (n = 4).

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RPB9 wild-type strain and five strains (Rev. #1, 3, 5, 6and 8) showed transcriptional error rate similar levelto the rpb9∆ cells (Fig. 4). These results suggest thatcertain gene mutation(s) which suppress the menadionesensitivity of the rpb9∆ cells could also restore thereduced transcriptional fidelity.

DiscussionStimulation of the cleavage activity intrinsic to RNA polymerase II contributes to transcriptional fidelity in yeast

The present study demonstrated that mutations thatcause a loss of S-II and Rpb9 cleavage stimulation activ-ity decreased transcriptional fidelity in yeast (Figs 1A and2A). Our present findings using S-II or Rpb9 mutationsindicate that stimulation of the RNA polymerase IIcleavage activity has an important role in maintainingtranscriptional fidelity in yeast.

Structure and function of S-II involved in maintaining transcriptional fidelity

S. cerevisiae S-II protein comprises 309 amino acid residues,and is divided into three distinct domains based on itsthree-dimensional structure (Fig. 5) (Wind & Reines2000). Domain I, located in the N-terminal region, isinvolved in interactions with other transcription factors(Saso et al. 2003; Wery et al. 2004). The other two regions,domain II and domain III, are essential for binding toRNA polymerase II and for both the cleavage and read-through stimulation activities, respectively (Nakanishiet al. 1995; Shimoaraiso et al. 1997; Awrey et al. 1998).X-ray structure analysis of the yeast RNA polymerase II-S-II complex revealed that domain III of S-II intrudesinto the pore of RNA polymerase II and approaches thecatalytic center of RNA polymerase II (Kettenbergeret al. 2003). In domain III, amino acid residues D290 andE291, which are conserved among eukaryotes, constitutethe “acidic hairpin” motif, and this motif is suggested tobe important for the nascent RNA cleavage stimulation.Biochemical studies also support the importance ofthese acidic residues for the stimulation of cleavage. Forexample, yeast D291, human D–E and D–E of GreB,which is a bacterial orthologue of S-II, are essential forstimulating cleavage in vitro (Jeon et al. 1994; Awrey et al.1998; Sosunova et al. 2003). In addition, D–E residuesare also conserved in DNA polymerases that are com-petent for the 3′→ 5′ exonuclease activity required forthe proofreading function (Morrison et al. 1991; Joyce& Steitz 1994). Mutations of these two amino acids in

DNA polymerases prevent the proofreading activity,resulting in elevated DNA mutation frequencies in vivo.These findings support that the cleavage stimulationactivity mediated by the D–E acidic residues of S-II iscritical for the proofreading activity during transcription.There has been no experimental evidence, however,that these two amino acids contribute to transcriptionalfidelity in yeast. In the present study, the Mt3 protein,which has mutations at amino acids E291 and R287 ofS-II, is unable to maintain transcriptional fidelity in yeast(Fig. 1A). In contrast, the Mt9 protein, which has a solemutation at R287, has no detectable defect in transcrip-tional fidelity, suggesting that the mutation at E291 isresponsible for the reduced transcriptional fidelity atleast when combined with mutation at R287 in yeast.

Figure 4 Menadione-resistant revertants for the rpb9∆ cells andtheir transcriptional fidelity. (A) Serial dilutions of menadione-resistant revertants (Rev. #1–8) for the rpb9∆ cells were spotted onto plates containing 25 µm menadione (indicated as “+”), andincubated at 30 °C. A plate image without the drug is also shown(indicated as “–”). (B) Transcriptional error rates of the revertantstrains. The error rate of the rpb9∆ strain was defined as 100%.Experiments were performed in duplicate (Rev. #1, #5–7,wild-type, and rpb9∆), triplicate (Rev. #3, 4 and 8), or tetraplicate(Rev. #2). Mean values with standard errors of the mean areshown.



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Therefore, we conclude that E291 has an important rolein maintaining transcriptional fidelity in yeast.

We found that cells expressing the Mt5 protein(K307A) showed no detectable defect in transcriptionalfidelity maintenance. According to the in vitro analysis inwhich truncated S-II proteins containing the C-terminalhalf of S-II (131–309 amino acid residues) are employed,the truncated S-II protein bearing K307A mutation isinactive or partially active in the read-through stimula-tion at 500 : 1 ratio to RNA polymerase II (Awrey et al.1998). In contrast, the truncated S-II bearing no pointmutation is active at 5 : 1 ratio to RNA polymerase II.Thus, the read-through stimulation activity of thetruncated form of the K307A protein is less than 1/100compared to that of the truncated S-II in vitro. The trun-cated form of the K307A protein would have similarread-through stimulation activity to the full-length formof the K307A protein Mt5, because the C-terminal halfof S-II is considered to be sufficient to promote read-through with efficiency nearly identical to the full-lengthS-II (Cipres-Palacin & Kane 1994; Awrey et al. 1998).Thus, the present result suggests that the transcriptionalresumption stimulation activity is not necessary fortranscriptional fidelity. We cannot exclude the possibil-ity that, unlike the truncated S-II bearing K307A muta-tion, the Mt5 protein might have the transcriptionalresumption stimulation activity due to its N-terminalhalf and this activity is involved in maintaining tran-scriptional fidelity.

Mt2-expressing cells showed higher error rate thanMt9-expressing cells although these two proteinsstimulate the RNA polymerase II cleavage activity to asimilar extent in vitro. At this moment, we do not haveany clear explanation for this contradiction on the basisof any experimental data. One possible explanation is thatthe cleavage stimulation activity of the Mt9 protein mightbe higher than that of the Mt2 protein although thesetwo proteins are classified into the same group for thein vitro cleavage stimulation activity (Awrey et al. 1998).

Involvement of S-II in Rpb9-mediated transcriptional fidelity

Table 1 summarizes the effects of S-II in preventingtranscriptional errors in yeast cells carrying the variousmutant RPB9 genes. The effect of S-II in preventingtranscriptional errors in the wild-type RPB9 cells(column “Wild-type”) was stronger than that in the RPB9null mutant cells (column “RPB9 null”). Thus, the effectof S-II in preventing transcriptional errors is affected bythe presence of the RPB9 gene. S-II exerts its transcrip-tional error avoidance function more effectively in cellsbearing the R91A, D94A, or ∆89-95 mutant RPB9 geneswhich encode proteins that have S-II dependent cleavagestimulation activity (Hemming & Edwards 2000) than inthe RPB9 null mutant cells. Meanwhile, the effect of S-IIwas not significantly enhanced in cells bearing the 1-47or ∆65-70 mutant RPB9 genes which encode proteins thatdo not have the cleavage stimulation activity, comparedwith that in the RPB9 null mutant cells. Therefore, theeffect of S-II in transcriptional error avoidance is likelyenhanced by Rpb9, and this Rpb9-mediated enhancementis accomplished by promoting the S-II cleavage stimulationactivity. Thus, modulation of the S-II cleavage stimu-lation activity by Rpb9 contributes to transcriptionalfidelity. In other words, the role of Rpb9 in maintainingtranscriptional fidelity is at least partially dependent onits ability to enhance the S-II cleavage stimulation.

S-II independent mechanism(s) for maintaining transcriptional fidelity mediated by Rpb9

Rpb9 comprises 122 amino acids and two zinc ribbondomains (Woychik et al. 1991). Rpb9 forms a “jaw”with Rpb1, a catalytic subunit of RNA polymerase II.Although Rpb9 is located close to S-II but does notdirectly interact with S-II (Fig. 4; Kettenberger et al.2003), Rpb9 functionally interacts with S-II in both thecleavage and read-through stimulation activities during

Table 1 Effect of S-II on transcriptional error avoidance in various RPB9 mutant strains

Strain RPB9 null Wild–type 1-47 ∆65-70 R91A D94A ∆89-95

1Effect of S-II 4.4 ± 0.3 7.8 ± 0.3 3.9 ± 1.1 2.7 ± 0.1 7.5 ± 1.0 8.5 ± 0.6 7.6 ± 0.72Cleavage – + – – + + +

1Effect of S-II on transcriptional error avoidance is defined as the ratio of the transcriptional error rate in the DST1 gene-absent background to that in the DST1 gene-present background. For example, transcriptional error rate in cells bearing wild-type RPB9 gene in the dst1∆ background was 7.8 times higher than that in cells bearing wild-type RPB9 gene in the DST1 background. Mean values with standard errors of the mean calculated from three independent experiments are shown.2The S-II dependent cleavage stimulation activity of the Rpb9 mutants is shown as described previously (Hemming & Edwards 2000).

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transcription elongation in vitro (Hemming & Edwards2000). In the present study, the Rpb9 activity thatenhances S-II cleavage stimulation activity significantlycontributed to transcriptional fidelity in vivo (Fig. 2A;Table 1). Rpb9 also contributes to transcriptionalfidelity in yeast cells in the absence of S-II (Fig. 3A).Nesser et al. recently reported that Rpb9 is importantfor maintaining transcriptional fidelity, and that Rpb9has more prominent effects in maintaining transcrip-tional fidelity than S-II. These findings indicate thatRpb9 is capable of maintaining transcriptional fidelity,at least in part, in an S-II independent manner. In otherwords, the roles of Rpb9 in transcriptional fidelityconsist of both S-II dependent (cleavage stimulation)and independent functions.

Our results obtained with the mutant RPB9 genes,∆65-70 and ∆89-95 were interpreted as follows. In theabsence of S-II, the ∆65-70 gene had a strong capacityto maintain transcriptional fidelity, as did the wild-typeRPB9 gene (Fig. 3A), because the ∆65-70 protein possessesthe S-II independent function for maintaining tran-scriptional fidelity. In contrast, in the presence of S-II,introduction of the ∆65-70 gene only partially restoredtranscriptional fidelity (Fig. 2A) due to the lack of S-IIdependent cleavage stimulation activity (Hemming &Edwards 2000). The ∆89-95 protein maintained tran-scriptional fidelity only in the presence of S-II, but itseffect was partial (Fig. 2A), as the ∆89-95 protein possesses

the S-II dependent cleavage stimulation (Hemming &Edwards 2000), but essentially lacks S-II independent activity.

It remains to be elucidated how Rpb9 maintainstranscriptional fidelity in an S-II independent manner.The ∆89-95 and ∆65-70 mutations might be good toolsfor elucidating the mechanism(s).

Enhanced sensitivity to oxidative stress and reduced transcriptional fidelity

Studies with several genes encoding mutant S-II andRpb9 proteins revealed that the mutations causingcompromised transcriptional fidelity in yeast also inducedoxidative stress-sensitive cell growth. Moreover, both thelack of the S-II/Rpb9-mediated cleavage stimulationand the lack of the S-II independent mechanism(s)exerted by Rpb9 resulted in oxidative stress-sensitive cellgrowth. One possibility is that the reduced transcriptionalfidelity in the DST1 or RPB9 disruptant was the causefor oxidative stress sensitivity, although it is also con-ceivable that S-II or Rpb9 might confer resistance tooxidative stress in yeast by relieving the transcriptionalarrest sites within genes essential for stress resistance.Oxidative stress causes generation of oxidized ribo-nucleotides, such as 8-oxo-GTP, in cells (Hayakawaet al. 1999). Mis-incorporation of 8-oxo-GTP into tran-scripts might lead to the production of proteins containingan amino acid substitution. Consistent with this notion,

Figure 5 Structure of S-II and Rpb9. Spatial relationships between S-II and Rpb9 proteins are presented. The mutation sites of themutant S-II and Rpb9 proteins used in this study are also shown. Domain I of S-II is not shown. This figure was prepared using the X-ray structure of the RNA polymerase II-S-II complex defined previously (Kettenberger et al. 2003). Other subunits of RNA polymeraseII besides S-II and Rpb9 are hidden.



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disruption of the MutT gene encoding 8-oxo-GTPasein E. coli causes elevated transcriptional errors probablydue to mis-incorporation of 8-oxo-GTP in vivo (Taddeiet al. 1997). To further clarify the relationship betweentranscriptional fidelity and oxidative stress sensitivity,it is a useful strategy to isolate revertants for the dst1∆or rpb9∆ cells. In this report, we isolated menadione-resistant revertants for the rpb9∆ cells (Fig. 4). We foundstrains whose transcriptional fidelity was restored (Rev. #2,4 and 7), suggesting a relationship between transcriptionalfidelity and oxidative stress-sensitive cell growth. Thesuppressor mutation(s) in the revertants isolated will beidentified. If the suppressor mutation(s) are located ingene(s) encoding transcription machinery including RNApolymerase II and transcription factors, the suppressormutation(s) might provide a clue to possible roles oftranscriptional fidelity maintenance in oxidative stressresistance. Mutation(s) in Rev. #1, 3, 5, 6 and 8 maysuppress menadione sensitivity by mechanism(s) notrelated to transcriptional fidelity. It is important to addresshow the mutation(s) restores reduced transcriptional fidelityand menadione sensitivity of the rpb9∆ cells.

Experimental proceduresYeast strains

All yeast strains used in this study are summarized in Table 2. Genedisruption of the DST1 gene was performed using a dst1::URA3or a dst1::HIS3 targeting vector. Vector structure and gene

disruption procedures were previously described (Ubukata et al.2002; Koyama et al. 2003).

Plasmids

Plasmids encoding the mutated Rpb9 proteins were constructedas follows. A fragment containing wild-type Rpb9 coding regionsand promoters was amplified by polymerase chain reaction usingyeast genomic DNA as a template with a pair of primers: 5′-GGAT-CAACTGGCCTTACATC-3′, located 0.4 kb upstream of thetranslation initiator ATG codon of the RPB9 gene, and 5′-ACT-CATGAAAACTGCGTCCT-3′, located at the TGA stop codonof the RPB9 gene. The amplified fragment was cloned at theSmaI site of the pYO324 plasmid (Ohya et al. 1991), and the NdeIsite was introduced immediately upstream of the start ATGcodon (converted from GCTATG to CATATG, the underlinedsequence is the NdeI site) to facilitate the vector construction pro-cedures. The wild-type Rpb9 coding regions on the plasmid werereplaced with the mutated Rpb9 protein coding regions obtainedby polymerase chain reaction. The fragments containing themutated Rpb9 protein coding regions and promoter regions wereexcised by digesting with NotI and SalI, followed by cloning intopYO326 (2 µ origin, harboring URA3 as a marker gene) vector(Sekiya-Kawasaki et al. 2002), which was digested with NotI andSalI. The resulting plasmids were used as expression vectors of themutated Rpb9 proteins.

The fidelity reporter plasmids harboring the wild-type (pWLac)or mutated lacZ (pMLac) in which a GAG triplet encodingglutamate-461 converted to TAG were previously described(Koyama et al. 2003). Briefly, the lacZ coding regions wereinserted into the downstream of the ADH1 promoter of thepACT2 plasmid (BD Biosciences; Lexington, KY).

Table 2 Yeast strains used in this study

Strain Genotype Reference

YPH499 MAT α ura3-52 lys2-801amber ade2-101ochre trp1-63 his3-∆ 200 leu2-∆1 Sikorski & Hieter 1989HKY01 MAT α ura3-52 lys2-801amber ade2-101ochre trp1-63 his3-∆ 200 leu2-∆1 dst1::URA3 Ubukata et al. 2002Mt1 MAT α ura3-52 lys2-801amber ade2-101ochre trp1-63 his3-∆ 200 leu2-∆1 dst1K242A/

Q243AUbukata et al. 2002

Mt2 MAT α ura3-52 lys2-801amber ade2-101ochre trp1-63 his3-∆ 200 leu2-∆1 dst1N252A/N255A/Q257A

Ubukata et al. 2002

Mt3 MAT α ura3-52 lys2-801amber ade2-101ochre trp1-63 his3-∆ 200 leu2-∆1 dst1R287Q/E291L Ubukata et al. 2002Mt4 MAT α ura3-52 lys2-801amber ade2-101ochre trp1-63 his3-∆ 200 leu2-∆1 dst1F296A Ubukata et al. 2002Mt5 MAT α ura3-52 lys2-801amber ade2-101ochre trp1-63 his3-∆ 200 leu2-∆1 dst1K307A Ubukata et al. 2002Mt6 MAT α ura3-52 lys2-801amber ade2-101ochre trp1-63 his3-∆ 200 leu2-∆1 dst1R200A Ubukata et al. 2002Mt7 MAT α ura3-52 lys2-801amber ade2-101ochre trp1-63 his3-∆ 200 leu2-∆1 dst1F269A Ubukata et al. 2002Mt9 MAT α ura3-52 lys2-801amber ade2-101ochre trp1-63 his3-∆ 200 leu2-∆1 dst1R287Q Ubukata et al. 2002BY4742 MAT α his3 ∆1 leu2 ∆ 0 lys2 ∆ 0 ura3 ∆ 0 Winzeler et al. 1999dst1 ∆ MAT α his3 ∆1 leu2 ∆ 0 lys2 ∆ 0 ura3 ∆ 0 dst1::KanMX Winzeler et al. 1999rpb9 ∆ MAT α his3 ∆1 leu2 ∆ 0 lys2 ∆ 0 ura3 ∆ 0 rpb9::KanMX Winzeler et al. 1999dst1 ∆ rpb9 ∆ MAT α his3 ∆1 leu2 ∆ 0 lys2 ∆ 0 ura3 ∆ 0 rpb9::KanMX dst1::HIS3 This study

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Fidelity assays

Yeast cells harboring pMLac or pWLac were cultured in SD mediumat 30 °C until the optical density at 600 nm (OD600) reachedapproximately 3.0 OD units. The cells were diluted to 0.1 ODunits, and incubated in SD medium containing 2% glucose untilthe OD600 reached 0.5–1.0 OD units. The cell lysis procedureand measurement of β-galactosidase activity were essentially aspreviously described. Protein concentrations were quantifiedby the Bradford method using bovine serum albumin as astandard.

Oxidant sensitivity assays

Oxidants used were menadione and hydrogen peroxide. Yeaststrains were cultivated at 30 °C in SD medium supplemented withappropriate nutrients until the OD600 reached approximately 2.8(early stationary phase) OD units (Figs 1–3), or 0.5 (mid-logphase) OD units (Fig. 4). The cultures were serially diluted sixfold,and 10 µL of each diluted culture was spotted on to SD agar platescontaining menadione, hydrogen peroxide, or no drug. Glucoseor galactose was used as sole carbon source in SD agar plates formenadione or hydrogen peroxide sensitivity assay, respectively.The plates were incubated at 30 °C for 4 days (menadione) or6 days (hydrogen peroxide). To evaluate survival, yeast cells werespread on SD agar plates containing either menadione or no drug.After incubation at 30 °C for 4 days, survival (%) was calculatedby the ratio of colony numbers that appeared in the presence ofmenadione to that in the absence of menadione.

Isolation of menadione-resistant revertants

The rpb9∆ cells were mutagenized with ethylmethane sulfonate(EMS) as previously described (Guthrie & Fink 1991). Aftermutagenesis, the EMS-treated cells were resuspended in SDmedium and cultured until early stationary phase. The cultureswere spread on to SD agar plate containing 25 µm menadione.The plates were incubated at 30 °C for several days, and menadione-resistant colonies were selected.

AcknowledgementsThis work was supported by grants from the Ministry ofEducation, Culture, Sports, Science and Technology of Japan,and the Japan Society for the Promotion of Science ( JSPS).H.K. is a postdoctoral JSPS Research Fellowship for YoungScientist.

ReferencesAwrey, D.E., Shimasaki, N., Koth, C., Weilbaecher, R., Olmsted,

V., Kazanis, S., Shan, X., Arellano, J., Arrowsmith, C.H., Kane,C.M. & Edwards, A.M. (1998) Yeast transcript elongationfactor (TFIIS), structure and function. II: RNA polymerasebinding, transcript cleavage, and read-through. J. Biol. Chem.273, 22595–22605.

Awrey, D.E., Weilbaecher, R.G., Hemming, S.A., Orlicky, S.M.,Kane, C.M. & Edwards, A.M. (1997) Transcription elongationthrough DNA arrest sites. A multistep process involving bothRNA polymerase II subunit RPB9 and TFIIS. J. Biol. Chem.272, 14747–14754.

Cipres-Palacin, G. & Kane, C.M. (1994) Cleavage of the nascenttranscript induced by TFIIS is insufficient to promote read-through of intrinsic blocks to elongation by RNA polymeraseII. Proc. Natl. Acad. Sci. USA 91, 8087–8091.

Erie, D.A., Hajiseyedjavadi, O., Young, M.C. & von Hippel, P.H.(1993) Multiple RNA polymerase conformations and GreA:control of the fidelity of transcription. Science 262, 867–873.

Exinger, F. & Lacroute, F. (1992) 6-Azauracil inhibition ofGTP biosynthesis in Saccharomyces cerevisiae. Curr. Genet. 22,9–11.

Fish, R.N. & Kane, C.M. (2002) Promoting elongation withtranscript cleavage stimulatory factors. Biochim. Biophys. Acta1577, 287–307.

Guthrie, C. & Fink, G.R. (1991) Guide to yeast genetics andmolecular biology. Methods Enzymol. 194, 1–863.

Hayakawa, H., Hofer, A., Thelander, L., Kitajima, S., Cai, Y.,Oshiro, S., Yakushiji, H., Nakabeppu, Y., Kuwano, M. &Sekiguchi, M. (1999) Metabolic fate of oxidized guanine ribo-nucleotides in mammalian cells. Biochemistry 38, 3610–3614.

Hemming, S.A. & Edwards, A.M. (2000) Yeast RNA polymeraseII subunit RPB9. Mapping of domains required for tran-scription elongation. J. Biol. Chem. 275, 2288–2294.

Hemming, S.A., Jansma, D.B., Macgregor, P.F., Goryachev, A.,Friesen, J.D. & Edwards, A.M. (2000) RNA polymerase IIsubunit Rpb9 regulates transcription elongation in vivo. J. Biol.Chem. 275, 35506–35511.

Hull, M.W., McKune, K. & Woychik, N.A. (1995) RNApolymerase II subunit RPB9 is required for accurate start siteselection. Genes Dev. 9, 481–490.

Izban, M.G. & Luse, D.S. (1992) The RNA polymerase II ternarycomplex cleaves the nascent transcript in a-3′→ 5′ directionin the presence of elongation factor SII. Genes Dev. 6, 1342–1356.

Jeon, C. & Agarwal, K. (1996) Fidelity of RNA polymerase IItranscription controlled by elongation factor TFIIS. Proc. Natl.Acad. Sci. USA 93, 13677–13682.

Jeon, C., Yoon, H. & Agarwal, K. (1994) The transcription factorTFIIS zinc ribbon dipeptide Asp-Glu is critical for stimulationof elongation and RNA cleavage by RNA polymerase II.Proc. Natl. Acad. Sci. USA 91, 9106–9110.

Joyce, C.M. & Steitz, T.A. (1994) Function and structure relation-ships in DNA polymerases. Annu. Rev. Biochem. 63, 777–822.

Kettenberger, H., Armache, K.J. & Cramer, P. (2003) Archi-tecture of the RNA polymerase II-TFIIS complex andimplications for mRNA cleavage. Cell 114, 347–357.

Koyama, H., Ito, T., Nakanishi, T., Kawamura, N. & Sekimizu,K. (2003) Transcription elongation factor S-II maintainstranscriptional fidelity and confers oxidative stress resistance.Genes Cells 8, 779–788.

Lange, U. & Hausner, W. (2004) Transcriptional fidelity andproofreading in Archaea and implications for the mechanismof TFS-induced RNA cleavage. Mol. Microbiol. 52, 1133–1143.



559

Morrison, A., Bell, J.B., Kunkel, T.A. & Sugino, A. (1991)Eukaryotic DNA polymerase amino acid sequence requiredfor 3′→ 5′ exonuclease activity. Proc. Natl. Acad. Sci. USA 88,9473–9477.

Nakanishi, T., Nakano, A., Nomura, K., Sekimizu, K. & Natori, S.(1992) Purification, gene cloning, and gene disruption ofthe transcription elongation factor S-II in Saccharomyces cerevi-siae. J. Biol. Chem. 267, 13200–13204.

Nakanishi, T., Shimoaraiso, M., Kubo, T. & Natori, S. (1995)Structure–function relationship of yeast S-II in terms of stimu-lation of RNA polymerase II, arrest relief, and suppression of6-azauracil sensitivity. J. Biol. Chem. 270, 8991–8995.

Natori, S., Takeuchi, K. & Mizuno, D. (1973) DNA-dependentRNA polymerase from Ehrlich ascites tumor cells. 3. Ribo-nuclease H and elongating activity of stimulatory factor S-II.J. Biochem. (Tokyo) 74, 1177–1182.

Nesser, N.K., Peterson, D.O. & Hawley, D.K. (2006) RNApolymerase II subunit Rpb9 is important for transcriptionalfidelity in vivo. Proc. Natl. Acad. Sci. USA 103, 3268–3273.

Ohya, Y., Goebl, M., Goodman, L.E., Petersen-Bjorn, S.,Friesen, J.D., Tamanoi, F. & Anraku, Y. (1991) Yeast CAL1 isa structural and functional homologue to the DPR1 (RAM) geneinvolved in ras processing. J. Biol. Chem. 266, 12356–12360.

Orlova, M., Newlands, J., Das, A., Goldfarb, A. & Borukhov, S.(1995) Intrinsic transcript cleavage activity of RNA poly-merase. Proc. Natl. Acad. Sci. USA 92, 4596–4600.

Reines, D. (1992) Elongation factor-dependent transcript short-ening by template-engaged RNA polymerase II. J. Biol. Chem.267, 3795–3800.

Reines, D., Chamberlin, M.J. & Kane, C.M. (1989) Transcriptionelongation factor SII (TFIIS) enables RNA polymerase II toelongate through a block to transcription in a human genein vitro. J. Biol. Chem. 264, 10799–10809.

Saso, K., Ito, T., Natori, S. & Sekimizu, K. (2003) Identificationof a novel tissue-specific transcriptional activator FESTA as aprotein that interacts with the transcription elongation factorS-II. J. Biochem. (Tokyo) 133, 493–500.

Sekimizu, K., Nakanishi, Y., Mizuno, D. & Natori, S. (1979)Purification and preparation of antibody to RNA polymeraseII stimulatory factors from Ehrlich ascites tumor cells. Bio-chemistry 18, 1582–1588.

Sekiya-Kawasaki, M., Abe, M., Saka, A., Watanabe, D., Kono, K.,Minemura-Asakawa, M., Ishihara, S., Watanabe, T. & Ohya, Y.(2002) Dissection of upstream regulatory components ofthe Rho1p effector, 1,3-β-glucan synthase, in Saccharomycescerevisiae. Genetics 162, 663–676.

Shaw, R.J. & Reines, D. (2000) Saccharomyces cerevisiae tran-scription elongation mutants are defective in PUR5 induction inresponse to nucleotide depletion. Mol. Cell. Biol. 20, 7427–7437.

Shimoaraiso, M., Nakanishi, T., Kubo, T. & Natori, S. (1997)Identification of the region in yeast S-II that defines speciesspecificity in its interaction with RNA polymerase II. J. Biol.Chem. 272, 26550–26554.

Shimoaraiso, M., Nakanishi, T., Kubo, T. & Natori, S. (2000)Transcription elongation factor S-II confers yeast resistanceto 6-azauracil by enhancing expression of the SSM1 gene.J. Biol. Chem. 275, 29623–29627.

Sikorski, R.S. & Hieter, P. (1989) A system of shuttle vectors andyeast host strains designed for efficient manipulation of DNAin Saccharomyces cerevisiae. Genetics 122, 19–27.

Sosunova, E., Sosunov, V., Kozlov, M., Nikiforov, V., Goldfarb,A. & Mustaev, A. (2003) Donation of catalytic residues toRNA polymerase active center by transcription factor Gre.Proc. Natl. Acad. Sci. USA 100, 15469–15474.

Taddei, F., Hayakawa, H., Bouton, M., Cirinesi, A., Matic, I.,Sekiguchi, M. & Radman, M. (1997) Counteraction by MutTprotein of transcriptional errors caused by oxidative damage.Science 278, 128–130.

Thomas, M.J., Platas, A.A. & Hawley, D.K. (1998) Tran-scriptional fidelity and proofreading by RNA polymerase II.Cell 93, 627–637.

Ubukata, T., Shimizu, T., Adachi, N., Sekimizu, K. & Nakanishi,T. (2002) Cleavage, but not read-through, stimulation activityis responsible for three biologic functions of transcriptionelongation factor S-II. J. Biol. Chem. 278, 8580–8585.

Van Mullem, V., Wery, M., Werner, M., Vandenhaute, J. &Thuriaux, P. (2002) The Rpb9 subunit of RNA polymerase IIbinds transcription factor TFIIE and interferes with the SAGAand elongator histone acetyltransferases. J. Biol. Chem. 277,10220–10225.

Wang, D. & Hawley, D.K. (1993) Identification of a-3′→ 5′exonuclease activity associated with human RNA polymeraseII. Proc. Natl. Acad. Sci. USA 90, 843–847.

Wery, M., Shematorova, E., Van Driessche, B., Vandenhaute, J.,Thuriaux, P. & Van Mullem, V. (2004) Members of the SAGAand Mediator complexes are partners of the transcriptionelongation factor TFIIS. EMBO J. 23, 4232–4242.

Wind, M. & Reines, D. (2000) Transcription elongation factorSII. Bioessays 22, 327–336.

Winzeler, E.A., Shoemaker, D.D., Astromoff, A., et al. (1999)Functional characterization of the S. cerevisiae genome by genedeletion and parallel analysis. Science 285, 901–906.

Woychik, N.A., Lane, W.S. & Young, R.A. (1991) Yeast RNApolymerase II subunit RPB9 is essential for growth at temperatureextremes. J. Biol. Chem. 266, 19053–19055.

Received: 22 August 2006Accepted: 28 January 2007

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Stimulation of RNA polymerase II transcript cleavage activity contributes to maintain transcriptional fidelity in yeast