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1 Department of Microbiology, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
2 New Product Research Laboratories III, Tokyo R&D Center, Daiichi Pharmaceutical Co., Ltd. Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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5' exonuclease activity to proofread the newly synthesized DNA (Joyce & Steitz 1994). By analogy, several studies have proposed that the 3' 5' nuclease activity intrinsic to RNA polymerases is involved in transcriptional proofreading (Erie et al. 1993; Jeon & Agarwal 1996; Thomas et al. 1998; Lange & Hausner 2004). Both prokaryotic and eukaryotic RNA polymerases possess the 3' 5' nuclease activity (Wang & Hawley 1993; Orlova et al. 1995). In E. coli, transcription elongation factors GreA and GreB activate the nuclease activity, leading to increased transcriptional fidelity in vitro (Erie et al. 1993). In eukaryotes, the 3' 5' nuclease activity of RNA polymerase II is stimulated by transcription elongation factor S-II (Izban & Luse 1992; Reines 1992). This process is further stimulated by Rpb9, a small subunit of RNA polymerase II (Awrey et al. 1997). There is no genetic evidence, however, to support the notion that stimulation of the 3' 5' nuclease activity intrinsic to the RNA polymerases contributes to maintain transcriptional fidelity in either prokaryotic or eukaryotic cells. Moreover, the physiologic relevance of the mechanisms that underlie the maintenance of transcriptional fidelity, including the elimination of oxidized nucleotides, the 3'5' nuclease activity of RNA polymerases, and the stimulation of this nuclease activity, remain uncertain.
Transcription elongation factor S-II/TFIIS was originally purified as a stimulatory protein of RNA polymerase II from mouse Ehrlich ascites tumor cells (Natori et al. 1973; Sekimizu et al. 1979). During RNA synthesis, RNA polymerase II is arrested by various transcriptional blocks, and in some cases, backtracks by several nucleotides along the template DNA. As a result, the 3'-end of the transcript dissociates from the catalytic center of RNA polymerase II (Fish & Kane 2002). Extensive biochemical studies revealed that S-II reactivates RNA polymerase II to read-through the arrest sites. When RNA polymerase II is arrested, the 3' 5' nuclease activity intrinsic to RNA polymerase II is stimulated by S-II to re-align the 3'-end of the transcript with the catalytic center, and then RNA polymerase II resumes transcription. Thus, stimulation of the 3' 5' nuclease activity by S-II is required for the S-II mediated read-through. This 3' 5' nuclease activity intrinsic to RNA polymerase II is designated "cleavage" activity. The cleavage 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 several mutant S-II proteins possess the cleavage stimulation activity, but do not promote read-through in vitro. Therefore, S-II is necessary not only for cleavage stimulation, but also for the transcriptional resumption step after the cleavage reaction in vitro. In other words, the read-through stimulation activity of S-II consists of two functions, stimulation of the cleavage reaction and transcriptional resumption after the cleavage reaction. Although the mechanism for the reactivation of RNA polymerase II after the cleavage reaction has not been characterized, several reports propose that a conformational change in RNA polymerase II might be necessary and that S-II is required for the change (Awrey et al. 1998).
In Saccharomyces cerevisiae, S-II is necessary for induction of the SSM1/SDT1 and the IMD2/PUR5 genes (Shaw & Reines 2000; Shimoaraiso et al. 2000). The SSM1 gene harbors transcriptional arrest sites. The IMD2 gene might contain the arrest sites, and mutations in the transcription elongation machinery render yeast cells defective in IMD2 gene induction. The cleavage, but not the read-through stimulation, of S-II is necessary for IMD2 gene induction in yeast (Ubukata et al. 2002). Thus, S-II mediated transcriptional resumption after cleavage is not essential for IMD2 gene induction. These results suggest that when RNA polymerase II reads through transcriptional arrest sites in yeast, a factor(s) other than S-II might be involved in the transcriptional resumption after the S-II mediated cleavage reaction (Ubukata et al. 2002).
Besides transcription arrest at specific sites on template DNA, RNA polymerase II also stops transcription temporarily when incorrect ribonucleotides are incorporated into the nascent transcripts (Thomas et al. 1998). In vitro studies demonstrated that S-II enhances the excision of these mis-incorporated nucleotides by stimulating the cleavage activity of RNA polymerase II (Jeon & Agarwal 1996). These results suggest that S-II contributes to transcriptional fidelity via transcriptional proofreading mediated by cleavage stimulation. Previously, we reported that 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 polymerase II cleavage activity is essential for the cellular function of S-II in transcriptional fidelity.
Rpb9, a small subunit of RNA polymerase II, functionally interacts with S-II (Hemming et al. 2000; Hemming & Edwards 2000). Rpb9 promotes the cleavage reaction by enhancing the response of RNA polymerase II to S-II. In other words, Rpb9 enhances the cleavage stimulation activity of S-II. Moreover, in the absence of Rpb9, S-II is unable to exert its read-through stimulation activity (Awrey et al. 1997). The RPB9 gene is dispensable for cell growth under usual culture conditions in S. cerevisiae, but loss of the RPB9 gene causes sensitivity to nucleotide-depleting drugs (6-azauracil and mycophenolic acid), which is similarly induced by DST1 gene disruption (Woychik et al. 1991; Exinger & Lacroute 1992; Nakanishi et al. 1992; Van Mullem et al. 2002). Recently, Rpb9 was shown to be involved in the maintenance of transcriptional fidelity in yeast (Nesser et al. 2006). It remains unclear whether the cleavage stimulation activity of Rpb9, which depends on S-II, serves to maintain transcriptional fidelity.
Here, we investigated whether the cleavage stimulation activity of S-II and Rpb9 contributes to transcriptional fidelity by using yeast strains bearing mutations on the DST1 and/or the RPB9 genes. Mutant strains that are defective in the maintenance of transcriptional fidelity have increased sensitivity to oxidative stress. We discuss the possibility that the mechanism underlying the maintenance of transcriptional fidelity conferred by stimulating RNA polymerase II cleavage activity is involved in the resistance to oxidative stress in yeast.
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Results |
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We previously reported that DST1 disrupted mutant yeast had reduced transcriptional fidelity and that the RNA polymerase II binding region in S-II is critical for the maintenance of transcriptional fidelity (Koyama et al. 2003). During transcription elongation in vitro, S-II stimulates the nascent RNA cleavage activity intrinsic to RNA polymerase II (Izban & Luse 1992). S-II also stimulates RNA polymerase II to read-through transcriptional arrest sites (Reines et al. 1989). This process comprises two distinct activities of S-II: stimulation of RNA cleavage and of transcriptional resumption after the cleavage reaction (Awrey et al. 1998). In this report, we call the latter activity "transcriptional resumption stimulation activity." To determine which of the two S-II activities is responsible for maintaining transcriptional fidelity in yeast, we examined transcriptional fidelity in yeast strains expressing a panel of point-mutated S-II proteins that have reduced cleavage and/or read-through stimulation activity in vitro (Awrey et al. 1998; Ubukata et al. 2002). Awrey et al. used truncated form of S-II proteins bearing point mutation(s) to evaluate the in vitro activities. The truncated S-II proteins contain the C-terminal half of the S-II protein (131–309 amino acid residues). In the present study, we used full-length S-II proteins bearing point mutation(s) which would have similar transcription activities to the truncated proteins bearing the same mutation(s), since the C-terminal half of S-II is sufficient to promote both cleavage and read-through with efficiencies nearly identical to the full-length S-II (Cipres-Palacin & Kane 1994; Nakanishi et al. 1995; Awrey et al. 1998). The activity scores of these S-II mutant proteins were defined according to Awrey et al. (Fig. 1A).
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cells (HKY01) was tenfold higher than that in the DST1 wild-type strain (YPH499). The error rate in Mt1, 4, 5, 6, 7, or 9-expressing cells was almost the same as that in YPH499, whereas Mt2 or Mt3-expressing cells had a much higher error rate than YPH499. Mt5 protein had reduced read-through stimulation activity, but did have strong cleavage stimulation activity. Therefore, the transcriptional resumption stimulation activity of S-II was compromised in this mutant protein. The error rate in the Mt5-expressing cells was as low as that in YPH499, suggesting that the S-II transcriptional resumption stimulation activity is not required for maintaining transcriptional fidelity. In contrast, Mt3 protein expression, which was defective in cleavage stimulation activity in vitro, failed to restore the high error rate in the DST1 null mutant strain. These results suggest that the S-II cleavage activity, but not the S-II transcriptional resumption stimulation activity, is responsible for maintaining transcriptional fidelity in yeast.
We previously reported that dst1 cells are sensitive to oxidative stress and have reduced transcriptional fidelity, and that the region of S-II protein essential for maintaining transcriptional fidelity coincides with the region that confers resistance to oxidative stress (Koyama et al. 2003). In the present study, we tested whether cells expressing point mutations of S-II, leading to reduced transcriptional fidelity, are sensitive to oxidative stress. As previously described, dst1 cells (HKY01) were more sensitive to the oxidants menadione and hydrogen peroxide than wild-type cells (YPH499) (Fig. 1B). Mt3-expressing cells were most sensitive to the oxidants. Mt2-expressing cells were also sensitive to the oxidants, whereas expression of the Mt1, 4, 5, or 9 proteins rescued the oxidant sensitivity in HKY01 to a level comparable to that in YPH499. Because Mt5-expressing cells are resistant to oxidative stress in spite of a defect in the transcriptional resumption stimulation activity, the reduction of the transcriptional resumption stimulation activity of S-II did not inevitably cause the oxidant sensitivity. In contrast, Mt3 protein expression, which was defective in the cleavage stimulation activity, failed to rescue the oxidants sensitivity. These results suggest that the cleavage stimulation activity of S-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 the nascent RNA cleavage activity intrinsic to RNA polymerase II, but only in the presence of S-II (Awrey et al. 1997). In other words, Rpb9 enhances the S-II cleavage stimulation activity. To further understand the contribution of the S-II cleavage stimulation activity to maintain transcriptional fidelity, we examined the involvement of Rpb9 in transcriptional fidelity. The transcriptional error rate in rpb9 cells was tenfold higher than that in RPB9 wild-type cells (BY4742), and the defect in transcriptional fidelity was restored by introducing a plasmid harboring the RPB9 gene (Fig. 2A). These results indicate that Rpb9 is involved in maintaining transcriptional fidelity in BY4742 yeast strains.
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Of the various Rpb9 mutant proteins, the 1-47 and 65-70 proteins are defective in both cleavage and read-through stimulation activities in vitro. These mutant proteins retain the activity of Rpb9 to select the accurate transcriptional start site in yeast (Hull et al. 1995; Hemming & Edwards 2000). The transcriptional error rate in rpb9 cells bearing plasmids encoding the 1-47 mutant gene was as high as that in rpb9 cells bearing the 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 R91A and D94A proteins were defective in the transcriptional resumption stimulation activity because these proteins lack the read-through stimulation activity, although they still 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 comparable to that in cells expressing wild-type Rpb9. The transcriptional error rate in cells expressing 89-95, the partial deletion mutant, which also possesses cleavage stimulation activity, but lacks read-through stimulation activity, was halfway between that in the rpb9 cells harboring the empty vector and that in the cells expressing wild-type Rpb9. These results demonstrate that the transcriptional resumption stimulation activity of Rpb9 is not essential for maintaining transcriptional fidelity, and that the cleavage stimulation activity is responsible for maintaining transcriptional fidelity.
To test whether loss of the cleavage stimulation activity of Rpb9 causes sensitivity to oxidative stress in yeast cells, we investigated colony formation of the rpb9 cells expressing various Rpb9 mutants in the presence of menadione. The rpb9 cells were much more sensitive to menadione than the RPB9 wild-type cells (BY4742), and the phenotype was suppressed by introducing plasmids harboring the wild-type RPB9 gene (Fig. 2B). Thus, Rpb9 confers resistance to oxidative stress in yeast cells. We next examined menadione sensitivity of cells expressing the mutant Rpb9 proteins defective in the cleavage stimulation activity (Fig. 2B,C). The rpb9 cells harboring plasmids containing the 1-47 or 65-70 genes were more sensitive to menadione than the wild-type RPB9 cells. On the other hand, the menadione sensitivity of the rpb9 cells was suppressed by introducing plasmids encoding the R91A or D94A mutant Rpb9 proteins, both of which retain cleavage stimulation activity, but are unable to enhance the read-through stimulation in vitro. Cells bearing the 89-95 gene were partially sensitive to menadione compared with the wild-type RPB9 cells. The cells bearing the 89-95 gene were apparently more resistant to menadione than the cells bearing the 65-70 gene in Fig. 2B, but was more sensitive in Fig. 2C. The observed variance is due to experimental fluctuation. Because the R91A and D94A proteins are considered to have defective transcriptional resumption stimulation activity, the lack of the transcriptional resumption stimulation activity of Rpb9 did not inevitably cause the menadione sensitivity. Furthermore, the suppression of menadione sensitivity by introduction of the 65-70 or 1-47 gene was slight or not significant, suggesting that the lack of cleavage stimulation activity of Rpb9 results in 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 transcriptional fidelity in yeast in an S-II independent manner (Nesser et al. 2006). This, together with our present results, suggests that Rpb9 maintains transcriptional fidelity via two different pathways, one that is dependent on the stimulation of cleavage by S-II and another that is S-II independent. To test this, we compared transcriptional fidelity in dst1 rpb9 double mutant cells with that in the dst1 cells. The transcriptional error rate in the dst1 rpb9 cells was higher than that in the dst1 cells (Fig. 3A), indicating that Rpb9 contributes to transcriptional fidelity in the absence of S-II.
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rpb9 cells carrying plasmids encoding a panel of mutant RPB9 genes. Cells bearing the 1-47 or 89-95 genes had a higher transcriptional error rate compared with cells bearing the wild-type RPB9 gene (Fig. 3A). In contrast, introduction of the R91A, D94A, or 65-70 genes restored the error rate to a level comparable to that in the wild-type RPB9 cells. These results indicate that the R91A, D94A, and 65-70 proteins maintain transcriptional fidelity in the absence of S-II. In other words, these three mutant proteins of Rpb9 contribute to maintain transcriptional fidelity in an S-II independent manner. Although the 65-70 protein had a slight effect on maintaining transcriptional fidelity in the presence of S-II, this protein contributed to transcriptional fidelity to a similar extent as the wild-type Rpb9 protein in the background of dst1 (Fig. 3A). Thus, the regions of Rpb9 necessary for transcriptional fidelity maintenance in the absence of S-II are distinct from the regions required in the presence of S-II. In contrast, although the 89-95 gene partially restored transcriptional fidelity in the presence of S-II (Fig. 2A), this capability of 89-95 was almost lost in the absence of S-II (Fig. 3A). These results indicate that the C-terminal region (down to amino acid residue 47 of Rpb9) is necessary for the function(s) of Rpb9 to maintain transcriptional fidelity in an S-II independent manner. Furthermore, of the C-terminal region, the amino acid residues 65–70, R91, and D94 are not required, but the amino acid residues 89–95 are important for maintaining transcriptional fidelity in the absence of S-II.
Next, we tested the menadione sensitivity of dst1 rpb9 cells carrying various mutant RPB9 genes. The dst1 rpb9 cells were more sensitive to menadione than the dst1 single mutant cells (Fig. 3B,C). This finding indicates that menadione sensitivity is caused not only by loss of the cleavage stimulation activity of Rpb9, but also by loss of another function(s) of Rpb9 that is independent of S-II. To determine critical regions of Rpb9 for menadione resistance in the absence of S-II, we tested whether menadione sensitivity is suppressed by introduction of the various mutant RPB9 genes. The dst1 rpb9 cells carrying the 1-47 or 89-95 genes were more sensitive to menadione than dst1 rpb9 cells carrying the wild-type RPB9 gene. Thus, in the absence of S-II, deletion of the C-terminal regions (down to amino acid residue 47) of Rpb9 causes menadione sensitivity. The amino acid residues 89-95 have a critically important role in menadione resistance. Meanwhile, when the gene encoding the R91A, D94A, or 65-70 proteins was introduced to dst1 rpb9 cells, menadione sensitivity was restored to the level of the wild-type RPB9 cells. Therefore, the amino acid residues of Rpb9 from 65 to 70, R91, or D94 are not essential for menadione resistance.
Isolation of revertants for the RPB9 gene disrupted yeast
Using various mutant RPB9 genes, we found that there is a good correlation between transcriptional fidelity and menadione sensitivity (Figs 2 and 3). To further assess the relationship between transcriptional fidelity and menadione sensitivity, we tried to isolate menadione-resistant revertants for the dst1 or the rpb9 cells. The rpb9 cells were mutagenized with ethylmethane sulfonate (EMS), and eight menadione-resistant revertant strains were obtained from the rpb9 cells (Fig. 4). We could not screen the dst1 cells for menadione-resistant revertant strains because of technical difficulties. Among the eight revertant strains isolated, three strains (Rev. #2, 4 and 7) showed reduced transcriptional error rate comparable with the RPB9 wild-type strain and five strains (Rev. #1, 3, 5, 6 and 8) showed transcriptional error rate similar level to the rpb9 cells (Fig. 4). These results suggest that certain gene mutation(s) which suppress the menadione sensitivity of the rpb9 cells could also restore the reduced transcriptional fidelity.
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Discussion |
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The present study demonstrated that mutations that cause a loss of S-II and Rpb9 cleavage stimulation activity decreased transcriptional fidelity in yeast (Figs 1A and 2A). Our present findings using S-II or Rpb9 mutations indicate that stimulation of the RNA polymerase II cleavage activity has an important role in maintaining transcriptional 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 its three-dimensional structure (Fig. 5) (Wind & Reines 2000). Domain I, located in the N-terminal region, is involved 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 to RNA polymerase II and for both the cleavage and read-through stimulation activities, respectively (Nakanishi et 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 intrudes into the pore of RNA polymerase II and approaches the catalytic center of RNA polymerase II (Kettenberger et al. 2003). In domain III, amino acid residues D290 and E291, which are conserved among eukaryotes, constitute the "acidic hairpin" motif, and this motif is suggested to be important for the nascent RNA cleavage stimulation. Biochemical studies also support the importance of these acidic residues for the stimulation of cleavage. For example, yeast D291, human D–E and D–E of GreB, which is a bacterial orthologue of S-II, are essential for stimulating cleavage in vitro (Jeon et al. 1994; Awrey et al. 1998; Sosunova et al. 2003). In addition, D–E residues are also conserved in DNA polymerases that are competent for the 3' 5' exonuclease activity required for the 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 stimulation activity mediated by the D–E acidic residues of S-II is critical for the proofreading activity during transcription. There has been no experimental evidence, however, that these two amino acids contribute to transcriptional fidelity in yeast. In the present study, the Mt3 protein, which has mutations at amino acids E291 and R287 of S-II, is unable to maintain transcriptional fidelity in yeast (Fig. 1A). In contrast, the Mt9 protein, which has a sole mutation at R287, has no detectable defect in transcriptional fidelity, suggesting that the mutation at E291 is responsible for the reduced transcriptional fidelity at least when combined with mutation at R287 in yeast. Therefore, we conclude that E291 has an important role in maintaining transcriptional fidelity in yeast.
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Mt2-expressing cells showed higher error rate than Mt9-expressing cells although these two proteins stimulate the RNA polymerase II cleavage activity to a similar extent in vitro. At this moment, we do not have any clear explanation for this contradiction on the basis of any experimental data. One possible explanation is that the cleavage stimulation activity of the Mt9 protein might be higher than that of the Mt2 protein although these two proteins are classified into the same group for the in 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 preventing transcriptional errors in yeast cells carrying the various mutant RPB9 genes. The effect of S-II in preventing transcriptional errors in the wild-type RPB9 cells (column "Wild-type") was stronger than that in the RPB9 null mutant cells (column "RPB9 null"). Thus, the effect of S-II in preventing transcriptional errors is affected by the presence of the RPB9 gene. S-II exerts its transcriptional error avoidance function more effectively in cells bearing the R91A, D94A, or 89-95 mutant RPB9 genes which encode proteins that have S-II dependent cleavage stimulation activity (Hemming & Edwards 2000) than in the RPB9 null mutant cells. Meanwhile, the effect of S-II was not significantly enhanced in cells bearing the 1-47 or 65-70 mutant RPB9 genes which encode proteins that do not have the cleavage stimulation activity, compared with that in the RPB9 null mutant cells. Therefore, the effect of S-II in transcriptional error avoidance is likely enhanced by Rpb9, and this Rpb9-mediated enhancement is accomplished by promoting the S-II cleavage stimulation activity. Thus, modulation of the S-II cleavage stimulation activity by Rpb9 contributes to transcriptional fidelity. In other words, the role of Rpb9 in maintaining transcriptional fidelity is at least partially dependent on its ability to enhance the S-II cleavage stimulation.
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Rpb9 comprises 122 amino acids and two zinc ribbon domains (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 not directly interact with S-II (Fig. 4; Kettenberger et al. 2003), Rpb9 functionally interacts with S-II in both the cleavage and read-through stimulation activities during transcription elongation in vitro (Hemming & Edwards 2000). In the present study, the Rpb9 activity that enhances S-II cleavage stimulation activity significantly contributed to transcriptional fidelity in vivo (Fig. 2A; Table 1). Rpb9 also contributes to transcriptional fidelity in yeast cells in the absence of S-II (Fig. 3A). Nesser et al. recently reported that Rpb9 is important for maintaining transcriptional fidelity, and that Rpb9 has more prominent effects in maintaining transcriptional fidelity than S-II. These findings indicate that Rpb9 is capable of maintaining transcriptional fidelity, at least in part, in an S-II independent manner. In other words, the roles of Rpb9 in transcriptional fidelity consist 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 the absence of S-II, the 65-70 gene had a strong capacity to maintain transcriptional fidelity, as did the wild-type RPB9 gene (Fig. 3A), because the 65-70 protein possesses the S-II independent function for maintaining transcriptional fidelity. In contrast, in the presence of S-II, introduction of the 65-70 gene only partially restored transcriptional fidelity (Fig. 2A) due to the lack of S-II dependent cleavage stimulation activity (Hemming & Edwards 2000). The 89-95 protein maintained transcriptional fidelity only in the presence of S-II, but its effect 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 maintains transcriptional fidelity in an S-II independent manner. The 89-95 and 65-70 mutations might be good tools for elucidating the mechanism(s).
Enhanced sensitivity to oxidative stress and reduced transcriptional fidelity
Studies with several genes encoding mutant S-II and Rpb9 proteins revealed that the mutations causing compromised transcriptional fidelity in yeast also induced oxidative stress-sensitive cell growth. Moreover, both the lack of the S-II/Rpb9-mediated cleavage stimulation and the lack of the S-II independent mechanism(s) exerted by Rpb9 resulted in oxidative stress-sensitive cell growth. One possibility is that the reduced transcriptional fidelity in the DST1 or RPB9 disruptant was the cause for oxidative stress sensitivity, although it is also conceivable that S-II or Rpb9 might confer resistance to oxidative stress in yeast by relieving the transcriptional arrest sites within genes essential for stress resistance. Oxidative stress causes generation of oxidized ribonucleotides, such as 8-oxo-GTP, in cells (Hayakawa et al. 1999). Mis-incorporation of 8-oxo-GTP into transcripts might lead to the production of proteins containing an amino acid substitution. Consistent with this notion, disruption of the MutT gene encoding 8-oxo-GTPase in E. coli causes elevated transcriptional errors probably due to mis-incorporation of 8-oxo-GTP in vivo (Taddei et al. 1997). To further clarify the relationship between transcriptional 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 found strains whose transcriptional fidelity was restored (Rev. #2, 4 and 7), suggesting a relationship between transcriptional fidelity and oxidative stress-sensitive cell growth. The suppressor mutation(s) in the revertants isolated will be identified. If the suppressor mutation(s) are located in gene(s) encoding transcription machinery including RNA polymerase II and transcription factors, the suppressor mutation(s) might provide a clue to possible roles of transcriptional fidelity maintenance in oxidative stress resistance. Mutation(s) in Rev. #1, 3, 5, 6 and 8 may suppress menadione sensitivity by mechanism(s) not related to transcriptional fidelity. It is important to address how the mutation(s) restores reduced transcriptional fidelity and menadione sensitivity of the rpb9 cells.
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Experimental procedures |
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All yeast strains used in this study are summarized in Table 2. Gene disruption of the DST1 gene was performed using a dst1::URA3 or a dst1::HIS3 targeting vector. Vector structure and gene disruption procedures were previously described (Ubukata et al. 2002; Koyama et al. 2003).
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Plasmids encoding the mutated Rpb9 proteins were constructed as follows. A fragment containing wild-type Rpb9 coding regions and promoters was amplified by polymerase chain reaction using yeast genomic DNA as a template with a pair of primers: 5'-GGATCAACTGGCCTTACATC-3', located 0.4 kb upstream of the translation initiator ATG codon of the RPB9 gene, and 5'-ACTCATGAAAACTGCGTCCT-3', located at the TGA stop codon of the RPB9 gene. The amplified fragment was cloned at the SmaI site of the pYO324 plasmid (Ohya et al. 1991), and the NdeI site was introduced immediately upstream of the start ATG codon (converted from GCTATG to CATATG, the underlined sequence is the NdeI site) to facilitate the vector construction procedures. The wild-type Rpb9 coding regions on the plasmid were replaced with the mutated Rpb9 protein coding regions obtained by polymerase chain reaction. The fragments containing the mutated Rpb9 protein coding regions and promoter regions were excised by digesting with NotI and SalI, followed by cloning into pYO326 (2 µ origin, harboring URA3 as a marker gene) vector (Sekiya-Kawasaki et al. 2002), which was digested with NotI and SalI. The resulting plasmids were used as expression vectors of the mutated Rpb9 proteins.
The fidelity reporter plasmids harboring the wild-type (pWLac) or mutated lacZ (pMLac) in which a GAG triplet encoding glutamate-461 converted to TAG were previously described (Koyama et al. 2003). Briefly, the lacZ coding regions were inserted into the downstream of the ADH1 promoter of the pACT2 plasmid (BD Biosciences; Lexington, KY).
Fidelity assays
Yeast cells harboring pMLac or pWLac were cultured in SD medium at 30 °C until the optical density at 600 nm (OD600) reached approximately 3.0 OD units. The cells were diluted to 0.1 OD units, and incubated in SD medium containing 2% glucose until the OD600 reached 0.5–1.0 OD units. The cell lysis procedure and measurement of ß-galactosidase activity were essentially as previously described. Protein concentrations were quantified by the Bradford method using bovine serum albumin as a standard.
Oxidant sensitivity assays
Oxidants used were menadione and hydrogen peroxide. Yeast strains were cultivated at 30 °C in SD medium supplemented with appropriate nutrients until the OD600 reached approximately 2.8 (early stationary phase) OD units (Figs 1–3), or 0.5 (mid-log phase) OD units (Fig. 4). The cultures were serially diluted sixfold, and 10 µL of each diluted culture was spotted on to SD agar plates containing menadione, hydrogen peroxide, or no drug. Glucose or galactose was used as sole carbon source in SD agar plates for menadione or hydrogen peroxide sensitivity assay, respectively. The plates were incubated at 30 °C for 4 days (menadione) or 6 days (hydrogen peroxide). To evaluate survival, yeast cells were spread on SD agar plates containing either menadione or no drug. After incubation at 30 °C for 4 days, survival (%) was calculated by the ratio of colony numbers that appeared in the presence of menadione 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). After mutagenesis, the EMS-treated cells were resuspended in SD medium and cultured until early stationary phase. The cultures were 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.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: sekimizu{at}mol.f.u-tokyo.ac.jp
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References |
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Received: 22 August 2006
Accepted: 28 January 2007
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