Rapamycin-induced Translational Derepression of GCN4 mRNA Involves a Novel Mechanism for Activation of the eIF2{alpha} Kinase GCN2*

Hiroyuki Kubota {ddagger} §, Tohru Obata ¶, Kazuhisa Ota {ddagger} || **, Takuma Sasaki ¶ || and Takashi Ito {ddagger} ** {ddagger}{ddagger}

From the {ddagger}Division of Genome Biology, Division of Experimental Therapeutics, and ||Center for the Development of Molecular Target Drugs, Cancer Research Institute, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-0934, and the **Institute for Bioinformatics Research and Development (BIRD), Japan Science and Technology Corporation (JST), 5-3 Yonbancho, Chiyoda-ku, Tokyo 102-0081, Japan

Received for publication, March 27, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
When starved for amino acids, Saccharomyces cerevisiae accumulates uncharged tRNAs to activate its sole eukaryotic initiation factor (eIF) 2{alpha} kinase GCN2. Subsequent phosphorylation of eIF2{alpha} impedes general translation, but translationally derepresses the transcription factor GCN4, which induces expression of various biosynthetic genes to elicit general amino acid control response. By contrast, when supplied with enough nutrients, the yeast activates the target of rapamycin signaling pathway to stimulate translation initiation by facilitating the assembly of eIF4F. A cross-talk was suggested between the two pathways by rapamycin-induced translation of GCN4 mRNA. Here we show that rapamycin causes an increase in phosphorylated eIF2{alpha} to translationally derepress GCN4. This increment is not observed in the cells expressing mammalian non-GCN2 eIF2{alpha} kinases in place of GCN2. It is thus suggested that rapamycin does not inhibit dephosphorylation of eIF2{alpha} but rather activates the kinase GCN2. This activation seems to require an interaction between the kinase and uncharged tRNAs, because rapamycin, similar to amino acid starvation, fails to induce eIF2{alpha} phosphorylation in the cells with GCN2 defective in tRNA binding. However, in contrast with amino acid starvation, rapamycin activates GCN2 without increasing the amount of uncharged tRNAs, but presumably by modifying the tRNA binding affinity of GCN2.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
In response to starvation for a single amino acid, the budding yeast Saccharomyces cerevisiae induces numerous biosynthetic genes not only for the starved amino acid but also for other non-starved ones. This response is designated as general amino acid control and has been extensively studied as a paradigm for eukaryotic gene regulation (1, 2). Consequently, a master gene activator GCN4 has been demonstrated to govern the concerted induction of a large group of biosynthetic genes (1, 2). Intriguingly, the synthesis of GCN4 itself is regulated mainly at the level of translation: amino acid starvation derepresses translation of GCN4 mRNA. This derepression is mediated by a unique mechanism that requires both the four short open reading frames (ORFs)1 in its 5'-leader region and a decrease in the ternary complex composed of eukaryotic initiation factor 2 (eIF2), GTP, and methionyl initiator-tRNA (1, 2, 3). The decrease is induced by phosphorylation of eIF2{alpha}, because it converts the GDP-bound eIF2 from a substrate to an inhibitor of eIF2B, the guanine nucleotide exchange factor for eIF2, thereby leading to a decline of eIF2-GTP.

The phosphorylation of eIF2{alpha} constitutes an evolutionarily conserved mechanism for eukaryotic translational control (3). In contrast with mammals bearing four eIF2{alpha} kinases, the budding yeast has the sole kinase GCN2, which is activated by accumulation of uncharged tRNAs due to amino acid starvation. GCN2 binds uncharged tRNAs via its histidyl-tRNA synthetase (His-RS)-related region. While GCN2 is activated by various stresses other than amino acid starvation, including purine deprivation, glucose limitation, high salinity, and exposure to an alkylating agent, the His-RS-related region has proven essential in all of the cases examined (2).

When supplied with enough nutrients, the yeast cells activate translation for cell growth and proliferation, in which the target of rapamycin (TOR) signaling pathway plays a central role (4, 5, 6). The TOR protein, a member of the phosphatidylinositol 3-kinase superfamily, is involved in regulation of various cellular processes, including transcription, stabilization of mRNAs, translation initiation and elongation, biogenesis of tRNAs and ribosome, turnover of nutrient transporters, protein kinase C signaling, and autophagy (4, 5). For the translation initiation, TOR proteins are known to facilitate the assembly of eIF4F, a heterotrimeric complex composed of eIF4A, -4E, and -4G (6). The eIF4F binds the 7-methylguanosine-containing cap structure via eIF4E to recruit mRNA to ribosome.

Since both general control and the TOR signaling play important roles in translational response to nutritional variation, it is likely that these pathways are coordinately regulated. We thus examined a possible cross-talk between the two pathways and obtained evidence that GCN2 is activated by rapamycin via a novel mechanism independent of an increment of uncharged tRNAs.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Strains—The strains used are W303H (MATa ade2–1 trp1–1 leu2–3 ura3–1 can1–100), WH1 (MATa ade2–1 trp1–1 leu2–3 ura3–1 can1–100 gcn1{Delta}::KanMX), WH2 (MATa ade2–1 trp1–1 leu2–3 ura3–1 can1–100 gcn2{Delta}::KanMX), WH3 (MATa ade2–1 trp1–1 leu2–3 ura3–1 can1–100 TOR1–1), WH9 (MATa ade2–1 trp1–1 leu2–3 ura3–1 can1–100 gcn2-S577A), and WH10 (MATa ade2–1 trp1–1 leu2–3 ura3–1 can1–100 gcn2-S577E).

Plasmids—The plasmid p180 is a low copy number reporter construct to monitor translational derepression of GCN4 (7). It encodes for an mRNA bearing GCN4-lacZ fusion ORF preceded by the entire 5'-leader region of GCN4 mRNA, which contains four upstream ORFs responsible for the derepression (1). The plasmid pYC is a centromeretype variant of pYES2, an expression vector using GAL1 promoter (Invitrogen). The pYC01 expressing GCN2 under a control of GAL1 promoter was constructed by gap-repair cloning of the ORF for GCN2. The pYC02 and pYC03, expressing heme-regulated inhibitor (HRI) and double-stranded RNA-activated protein kinase (PKR), were constructed by transferring the SphI-KpnI fragment of p1246 and p1420 to pYC, respectively (8). The pYC04 bearing gcn2-m2 allele (9) was constructed from pYC01 by site-directed mutagenesis. The WH2 cells bearing these eIF2{alpha} kinase plasmids were cultivated in SCR (synthetic complete medium containing raffinose instead of glucose) lacking uracil and shifted to SCR – Ura – His + 20 mM 3-aminotriazole (3AT) or SCR – Ura + 200 ng/ml rapamycin.

Reporter Assay for Translational Derepression of GCN4 mRNA—The yeast cells bearing p180 were treated with either 200 ng/ml rapamycin or 20 mM 3AT in SC – Ura or SC – Ura – His, respectively, and assayed for {beta}-galactosidase as described previously (10, 11).

Immunoblotting Analysis of eIF2{alpha}Immunoblotting analysis of eIF2{alpha} was performed using an anti-eIF2{alpha} peptide antibody and the one specific to eIF2{alpha} phosphorylated at serine 51 (eIF2{alpha}[pS51]) (BIOSOURCE) as described previously (11). Isoelectric focusing gel electrophoresis was performed as described (12). Immunoblots were developed using a kit for enhanced chemiluminescence (ECL, Amarsham Biosciences).

Reverse Transcription-PCR—Isolation of total RNAs, reverse transcription, and PCR were performed as described previously (13).

Analysis of tRNAs—Aminoacylated tRNAs were analyzed as described (14). Briefly, the yeast cells were disrupted by vigorous shaking with glass beads in AE buffer (50 mM sodium acetate (pH 5.3), 10 mM EDTA) at 4 °C. Total RNAs were extracted with AE-saturated phenol and precipitated with ethanol. Deacylated tRNAs were prepared by heating at 75 °C in 0.1 M Tris-HCl (pH 8.0) for 5 min. Total RNAs (10 µg) were separated at 4 °C by electrophoresis on a 10% polyacrylamide gel containing 8 M urea in 100 mM sodium acetate buffer (pH 5.0). Separated RNAs were blotted onto Hybond N+ membrane (Amersham Biosciences) and hybridized with radiolabeled probe for each tRNA species.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Rapamycin Derepresses Translation of GCN4 mRNA—To test a possible cross talk between general control response and the TOR signaling, we examined the effect of rapamycin, a specific inhibitor of TOR proteins, on translational derepression of GCN4 mRNA. To monitor the derepression, we employed the p180 reporter construct bearing the 5'-leader region of GCN4 mRNA followed by lacZ ORF (7). When the yeast cells harboring p180 were treated with 3AT, an inducer of histidine starvation, {beta}-galactosidase activity was induced as described previously (7, 11) (Fig. 1A). We treated the same strain with rapamycin, whose action was confirmed in both wild and TOR1–1 cells by monitoring mRNAs for DAL4 and RPL4A, known to be induced and suppressed, respectively, by rapamycin in a TOR-dependent manner (Fig. 1B). As shown in Fig. 1A, an increase was observed in {beta}-galactosidase activity upon rapamycin treatment. Furthermore, we found that the cells bearing TOR1–1 allele induce the reporter in response to 3AT but not to rapamycin (Fig. 1A). These results indicate that the TOR signaling pathway is involved in modulation of GCN4 translation. Rapamycin-induced derepression of GCN4 mRNA was independently reported by others during the course of this work (15).



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FIG. 1.
Rapamycin-induced derepression of GCN4 translation and increment of eIF2{alpha}[pS51]. A, the W303H (wild type) and WH3 (TOR1-1) cells bearing the GCN4-lacZ reporter plasmid p180 were grown to mid logarithmic phase in SC – Ura, shifted to SC – Ura – His + 20 mM 3AT or SC – Ura + 200 ng/ml rapamycin, and assayed for {beta}-galactosidase activity at the time points indicated. B, reverse transcription-PCR was performed for ACT1, DAL4, and RPL4A using total RNAs isolated from W303H (wild type) and WH3 (TOR1--1) cells cultivated in the absence or presence of rapamycin. C, the phosphorylation of eIF2{alpha} was examined by immunoblotting using an anti-eIF2{alpha}[pS51] antibody in the W303H (wild), WH2 (gcn2{Delta}), WH1 (gcn1{Delta}), and WH3 (TOR1-1) cells treated with control vehicle (left), rapamycin (middle), or 3AT (right) for 2 h. Total amount of eIF2{alpha} was examined using an anti-eIF2{alpha} antibody in immunoblot. Translational derepression of GCN4 mRNA was measured as above.

 

Rapamycin-induced Translation of GCN4 mRNA Requires GCN2-mediated Phosphorylation of eIF2{alpha}Translational derepression of GCN4 mRNA does not always require the eIF2{alpha} kinase GCN2; several alternative mechanisms independent of GCN2 have been reported. For instance, a shift from amino acid-rich to minimal medium was shown to induce GCN4 translation independently of GCN2 (16). The reduction in amount or impairment in base modification of the initiator tRNA also leads to GCN2-independent derepression of GCN4 translation (1, 17). Similarly, overexpression of the RNA component of RNase MRP or tRNA pseudouridine 55 synthetase results in GCN4 derepression without activating GCN2 (18, 19). More recently, fusel alcohols such as butanol were shown to translationally induce GCN4 in a GCN2-independent manner (20).

It is thus important to examine whether rapamycin-induced GCN4 translation accompanies an increase in phosphorylated eIF2{alpha}. We performed an immunoblot analysis using an antibody specific to eIF2{alpha}[pS51]. The results indicated that rapamycin, similar to 3AT, induces a significant increase in eIF2{alpha}[pS51], but, importantly, does not affect total eIF2{alpha} levels (Fig. 1C). Judging from these results and previous studies on 3AT-treated cells (1, 2), we assume that ~50% of eIF2{alpha} can be phosphorylated by rapamycin-treatment. Interestingly, rapamycin, but not 3AT, failed to increase eIF2{alpha}[pS51] in the cells bearing TOR1–1 allele, which we had shown to be incapable of derepressing translation of GCN4 mRNA in response to rapamycin (Fig. 1C).

We next intended to know whether the observed increase in eIF2{alpha}[pS51] is required for the rapamycin-induced translation of GCN4 mRNA. For this purpose, we examined the yeast cells deleted for GCN2, because the gcn2{Delta} cell contains no eIF2{alpha} kinase activity and thus lacks eIF2{alpha}[pS51] (Fig. 1C). As shown in Fig. 1C, rapamycin, similar to 3AT, totally failed to induce {beta}-galatosidase activity in the gcn2{Delta} cells.

To further confirm the importance of the phosphorylation, we examined a strain deleted for GCN1, which encodes a GCN2-binding protein required for in vivo activation of the eIF2{alpha} kinase (10, 11, 21, 22). The gcn1{Delta} cells contain GCN2 in an amount comparable with that in the wild type cells, but fail to activate its kinase activity (8). As shown in Fig. 1C, the gcn1{Delta} cells could not induce the reporter activity upon rapamycin treatment. While a novel mode of GCN2 action independent of eIF2{alpha} phosphorylation was recently proposed (23), rapamycin-induced GCN4 translation requires the eIF2{alpha} kinase activity.

Neither Histidine Starvation nor Rapamycin Inhibits Dephosphorylation of eIF2{alpha}[pS51]—Theoretically, an increment of eIF2{alpha}[pS51] can be achieved not only by activation of GCN2 but also by impeded dephosphorylation of eIF2{alpha}[pS51]. Involvement of both mechanisms would be also plausible. We thus intended to examine a possible role for compromised dephosphorylation in the increment of eIF2{alpha}[pS51] induced by amino acid starvation and rapamycin. For this purpose, we generated strains lacking GCN2 but instead expressing a mammalian non-GCN2 eIF2{alpha} kinase, namely HRI or PKR (3). While overexpression of these eIF2{alpha} kinases was previously reported to induce a slow growth phenotype (8), our gcn2{Delta} strain expressing HRI from a single copy vector displays no apparent growth defect in a raffinose medium. On the other hand, the growth of the PKR-expressing strain was impaired. While the expression of these kinases did not affect total eIF2{alpha} levels (Ref. 8 and data not shown), both strains showed an induced level of eIF2{alpha}[pS51] comparable with that in the parental GCN2 cells subject to histidine starvation or rapamycin treatment (Fig. 2). These results suggest that the overexpressed eIF2{alpha} kinases are somehow disregulated and hence partly activated, as also shown in the previous study (8).



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FIG. 2.
Effects of histidine starvation and rapamycin on eIF2{alpha}[pS51] phosphorylated by GCN2, HRI, and PKR. The WH2 (gcn2{Delta}) cells bearing pYC01, pYC02, pYC03, each expressing GCN2, HRI, or PKR, respectively, were grown in SCR – Ura to mid logarithmic phase and shifted to SCR – Ura – His + 20 mM 3AT or SCR – Ura + 200 ng/ml rapamycin. Following cultivation for 2 h in the presence of the drugs, the amount of eIF2{alpha}[pS51] was examined by immunoblotting.

 

If amino acid starvation leads to inhibition of eIF2{alpha}[pS51] phosphatase(s), the steady-state level of eIF2{alpha}[pS51] is expected to increase even in the cells expressing the non-GCN2 kinases that are not activated by amino acid starvation (24, 25). We made the HRI-expressing cells starved for histidine and examined the amount of eIF2{alpha}[pS51]. In contrast with GCN2-expressing cells, histidine starvation did not increase eIF2{alpha}[pS51] in the HRI-expressing cells (Fig. 2). We also examined the PKR-expressing cells to find no change in the amount of eIF2{alpha}[pS51] (Fig. 2). It thus seems that amino acid starvation does not affect dephosphorylation of eIF2{alpha}[pS51].

We next examined the effect of rapamycin on dephosphorylation of eIF2{alpha}[pS51] using the same strains. We failed to detect any noticeable change in the amount of eIF2{alpha}[pS51] in the rapamycin-treated HRI-expressing cells (Fig. 2). Similarly, the PKR-expressing cells failed to increase eIF2{alpha}[pS51] upon rapamycin treatment (Fig. 2). While no evidence has ever been provided for or against rapamycin-induced modulation of HRI or PKR activity, it is highly unlikely that rapamycin affects both the kinases and phosphatase so finely as to keep the steady-state level of eIF2{alpha}[pS51] unaffected. It is thus strongly suggested that rapamycin, similar to amino acid starvation, does not inhibit dephosphorylation of eIF2{alpha}[pS51].

Rapamycin-induced Activation of GCN2 Requires Binding to Uncharged tRNAs—The results described above indicate that rapamycin induces an increase in eIF2{alpha}[pS51] via activation of the kinase GCN2. To be activated by amino acid starvation and other various stimuli, GCN2 has to bind uncharged tRNA via its His-RS-like domain (2, 9). We thus examined whether rapamycin-induced GCN2 activation requires an interaction with tRNA using gcn2-m2 mutant defective in tRNA binding (9). As shown in Fig. 3, the gcn2-m2 cells, maintaining a comparable eIF2{alpha} level to wild type cells (Ref. 9 and data not shown), respond neither to histidine starvation nor to rapamycin. Thus, the activation of GCN2 by rapamycin, like that by amino acid starvation, requires an interaction of the kinase with uncharged tRNA.



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FIG. 3.
Effects of histidine starvation and rapamycin on eIF2{alpha}[pS51] in the cells with gcn2-m2 allele defective in tRNA binding. The WH2 (gcn2{Delta}) cells bearing pYC01 (GCN2) or pYC04 (gcn2-m2) were grown to mid logarithmic phase in SCR – Ura, shifted to SCR – Ura – His + 20 mM 3AT or SCR – Ura + 200 ng/ml rapamycin, and examined for eIF2{alpha}[pS51] by immunoblotting.

 

Rapamycin Activates GCN2 without Increasing Uncharged tRNAs—Confirming the requirement for tRNA binding, we next examined whether rapamycin induces an increase in the amount of uncharged tRNAs. Total tRNAs were isolated from histidine-starved and rapamycin-treated cells under a cooled acidic condition so as to minimize deacylation and analyzed by acid-urea polyacrylamide gel electrophoresis. In this electrophoresis, aminoacylated tRNAs display more retarded mobility than uncharged ones, which were obtained through deacylation by a heat treatment under an alkaline condition (Fig. 4). As expected, 3AT-treatment facilitated deacylation of tRNA for histidine to prove the principle of the assay (Fig. 4).



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FIG. 4.
Effects of histidine starvation and rapamycin on aminoacylation of tRNAHis. The W303H cells were grown to mid logarithmic phase and shifted to SC – His + 20 mM 3AT or YPAD (1% yeast extract, 2% peptone, 2% glucose, 0.004% adenine) containing 200 ng/ml rapamycin. Total RNAs were isolated at the indicated time points under a cooled acidic condition to prevent deacylation, resolved by acid-urea polyacrylamide gel electrophoresis, transferred to a nylon membrane, and hybridized with tRNAHis-specific probe. Phosphorylation of eIF2{alpha} was examined by immunoblotting as above. The histidine-starved cells were replenished with histidine in the absence or presence of rapamycin (labeled as His and His + rap in the left panel) and examined for tRNAHis and eIF2{alpha}[pS51].

 

Replenishment of the 3AT-treated cells with histidine induced a rapid aminoacylation of the tRNA, thereby ceasing GCN2 activation to decline eIF2{alpha}[pS51]. Intriguingly, the aminoacylation induced by refeding of the starved amino acid was observed even in the cells pretreated with rapamycin. It thus seems that rapamycin does not affect de novo aminoacylation of tRNA.

We next examined tRNAs isolated from the cells treated with rapamycin in a rich medium. Rapamycin-treatment increased eIF2{alpha} phosphorylation but failed to induce no apparent change in electrophoretic pattern of bulk tRNAs: most displayed more retarded mobility than alkaline-treated ones, indicative of aminoacylated states (data not shown). Such constitutive aminoacylation was more clearly demonstrated by visualization of specific tRNA on Northern blot (Fig. 4). Therefore, in contrast with amino acid starvation, rapamycin can activate GCN2 without increasing uncharged tRNAs.

Ser-577 Plays a Role in Rapamycin-induced Activation of GCN2—In the context described above, it is quite intriguing to note that phosphorylation of GCN2 at Ser-577 was recently reported to reduce its binding affinity to uncharged tRNAs (26). We thus examined gcn2-S577A mutant cells to know whether this residue plays a role in rapamycin-induced activation of GCN2. As expected, the mutant cells showed a significantly higher basal GCN2 activity than its parental cells (Fig. 5). Notably, rapamycin failed to further derepress GCN4 translation in the mutant cells (Fig. 5). The cells bearing gcn2-S577E allele, which had been shown to only partly mimic Ser-577-phosphorylated GCN2 (26), also displayed a similar defective response to rapamycin (Fig. 5). It is thus likely that dephosphorylation of this residue is involved in rapamycin-induced GCN2 activation.



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FIG. 5.
Roles for Ser-577 in rapamycin-induced activation of GCN2. The W303H (wild type), WH9 (gcn2-S577A), and WH10 (gcn2-S577E) cells bearing the GCN4-lacZ reporter plasmid p180 were grown to mid logarithmic phase in SC – Ura, shifted to SC – Ura – His + 20 mM 3AT or SC – Ura + 200 ng/ml rapamycin for 2 h, and assayed for {beta}-galactosidase activity.

 

Conclusions—Here we demonstrated that rapamycin, similar to amino acid starvation, activates the eIF2{alpha} kinase GCN2 in an uncharged tRNA-dependent manner (Fig. 3), with constitutive dephosphorylation of eIF2{alpha}[pS51] (Fig. 2), to increase the amount of eIF2{alpha}[pS51], thereby leading to translational derepression of GCN4 mRNA (Fig. 1). However, in contrast with amino acid starvation, rapamycin can do so without increasing the amount of uncharged tRNAs (Fig. 4), but presumably by acting via dephosphorylation of Ser-577 to increase its tRNA-binding affinity (Fig. 5). Further pursuit of the mechanism for TOR-mediated inhibition of GCN2 would help us better understand not only the cross-talk between the two important pathways for nutritional stress response but the mode of action for rapamycin and its derivatives, which are currently used as immunosuppressants and potential anti-cancer drugs.


    FOOTNOTES
 
* This work was supported in part by grant-in-aid for Scientific Research on Priority Areas (C) "Genome Biology" from the Ministry of Education, Culture, Sports, Science and Technology of Japan and grant-in-aid for Scientific Research (B) from Japan Society for Promotion of Science (JSPS). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Recipient of postdoctoral fellowship from JSPS. Back

{ddagger}{ddagger} To whom correspondence should be addressed: Division of Genome Biology, Cancer Research Inst., Kanazawa University, 13-1 Takaramachi, Kanazawa 920-0934, Japan. Tel.: 81-76-265-2726; Fax: 81-76-234-4508; E-mail: titolab{at}kenroku.kanazawa-u.ac.jp.

1 The abbreviations used are: ORF, open reading frame; eIF, eukaryotic initiation factor; His-RS, histidyl-tRNA synthetase; TOR, target of rapamycin; HRI, heme-regulated inhibitor; PKR, double-stranded RNA-activated protein kinase; SCR, synthetic complete medium containing 2% raffinose; 3AT, 3-aminotriazole; SC, synthetic complete medium; eIF2{alpha}[pS51], eIF2{alpha} phosphorylated at Ser-51. Back


    ACKNOWLEDGMENTS
 
We thank A. G. Hinnebusch (National Institutes of Health) for generous gifts of plasmids p1246 and p1420.



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 ABSTRACT
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