From the Institut für Mikrobiologie und Genetik, Abteilung für Molekulare Mikrobiologie, Georg-August-Universität, Grisebachstrasse 8, D-37077 Göttingen, Germany
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ABSTRACT |
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In Saccharomyces cerevisiae the GCN4 gene encodes the transcriptional activator of the "general control" system of amino acid bioynthesis, a network of at least 12 different biosynthetic pathways. We characterized the consequences of the general control response upon the signal "amino acid starvation" induced by the histidine analogue 3-aminotriazole with respect to Gcn4p levels in more detail. Therefore, we established test systems to monitor the time course of different parameters, including GCN4 mRNA, Gcn4 protein, Gcn4p DNA binding activity, as well as Gcn4p transactivation ability. We observed a biphasic response of Gcn4p activity in the cell. At first, translation of GCN4 mRNA is induced within 20 min after switch to starvation conditions. However, an additional increase in GCN4 transcript steady state level was observed, leading to an additional second phase of GCN4 expression after 3-4 h of starvation. The DNA binding activity of Gcn4p, as well as the ability to activate transcription of target genes, correlate with the amount of Gcn4 protein in the cell, suggesting that under the tested conditions there is no additional regulation of DNA binding or transactivation ability of Gcn4p, respectively.
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INTRODUCTION |
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The survival of cells in their natural environment is dependent on their ability to adapt rapidly to occurring changes. For example, yeast cells must respond to changes in the availability of amino acids to survive in nature. In yeast, complex biological mechanisms have evolved to ensure a sufficient supply of amino acids within the cell. Starvation of the yeast Saccharomyces cerevisiae for any of several amino acids causes a coordinated derepression of genes encoding enzymes involved in more than 10 different unlinked amino acid biosynthetic pathways, as well as of amino acid tRNA synthetases (1, 2), and enzymes for purine biosynthesis (3). This complex interplay between numerous control mechanisms is known as "general amino acid control" (4).
Under laboratory conditions, the general control response is not inducible by culturing cells on minimal medium (4). Because of the high basal expression levels of genes involved in amino acid biosynthesis, the ability of yeast to synthesize at least one of the amino acids must be impaired. A common approach to impose amino acid limitation on S. cerevisiae is to cultivate yeast cells in the presence of amino acid analogues, as 3-aminotriazole (3-AT)1 or 5-methyltryptophan. The histidine analogue 3-AT is a competitive inhibitor of the imidazole glycerolphosphate dehydratase, an enzyme involved in histidine biosynthesis (5). Addition of 3-AT to exponentially growing yeast culture leads to insufficient supply of the cell with histidine which results in the activation of the general amino acid control (6).
The general control is a multifaceted regulatory mechanism that draws upon several different strategies to control gene expression. Gcn4p is the transcriptional activator of gene expression in this system. It enables the yeast cell to stimulate the expression of at least 40 different genes. Gcn4p is a member of the basic leucine zipper family (7) and binds directly as a homodimer to a conserved regulatory region of its target genes (8). This DNA sequence required for specific binding of Gcn4p is well defined and consists of the symmetric nucleotide motive TGA(C/G)TCA (9).
The rate of Gcn4p synthesis itself is regulated by the availability of
amino acids. Under nonstarvation conditions the GCN4 mRNA is poorly translated due to the negative effects of the
translation of four small upstream open reading frames (uORFs) present
in its 5'-untranslated region (10, 11). When cells are grown under
conditions of severe amino acid limitation, an elevation in the
cellular amount of Gcn4p is accomplished through an increased translation of GCN4 mRNA. A deletion of all four uORFs
(12) or point mutations in their four ATG start codons (13) results in
an increased translation of GCN4 mRNA. The model for
this translational control is that the four uORFs in the leader of
GCN4 mRNA restrict the flow of scanning ribosomes from
the cap site to the GCN4 initiation codon. When amino acids
are abundant, ribosomes translate the first uORF and reinitiate at one
of the remaining uORFs in the leader, after which they dissociate from
the mRNA. Under conditions of amino acid starvation, many ribosomes
which have translated uORF1 fail to reinitiate at uORFs 2 to 4 and
utilize the GCN4 start codon instead. The failure to
reinitiate at uORFs 2 to 4 in starved cells results from a reduction
in the GTP-bound form of eIF-2 that delivers charged initiator
tRNAiMet to the ribosome. When the levels of
eIF-2·GTP·Met-tRNAiMet ternary
complexes are low, many ribosomes will not rebind this critical
initiation factor following translation of uORF1 until after scanning
past uORF4, but before reaching GCN4 (14). It has been
supposed that under amino acid starvation conditions, uncharged tRNAs
accumulate and stimulate Gcn2p protein kinase activity by interacting
with its histidyl-tRNA synthetase-related domain (15). Gcn2p
phosphorylates the -subunit of eIF-2 (16). Phosphorylation of eIF-2
reduces the amount of active eIF-2 available for ternary complex
formation by inhibiting eIF-2B, the guanine nucleotide exchange factor.
The resulting low level of eIF-2·GTP and ternary complex formation
diminishes the rate at which initiation complexes are reassembled.
An additional regulation mechanism for the general control system is the regulation by protein degradation of Gcn4p. Pulse-chase experiments showed that Gcn4p is highly unstable, with a half-life of about 5 min under nonstarvation conditions (17). This degradation is inhibited under conditions of starvation for amino acids. Up to now nothing is known about other regulation mechanisms of GCN4 gene expression or Gcn4 protein itself.
The rapid turnover of Gcn4p may be the reason that Gcn4p activity could hardly be detected in yeast cells. Virtually all biochemical analyses of Gcn4p function have used Gcn4p produced by recombinant techniques, for example, analyses of GCN4 regulation in vivo have used GCN4::lacZ fusion genes. Here we have characterized in detail the consequences of the general control response with respect to GCN4 mRNA and Gcn4p levels. We have examined several parameters of the changes of Gcn4p levels when exponentially growing cells were shifted from growth in minimal medium to amino acid starvation medium. Test systems were developed in order to monitor the time course of various steps of this induction process: (a) GCN4 mRNA steady state levels were determined. (b) Gcn4 protein was detected using a specific antibody raised against the DNA-binding domain of Gcn4p. (c) An assay to detect Gcn4p in a gel retardation experiment using cellular extracts was developed. (d) A highly sensitive lacZ fusion gene as efficient target of the general control was constructed carrying multiple cassettes of the Gcn4p recognition element in its promoter to monitor the trans-activation rate of the Gcn4 protein. These assays were performed with both a strain harboring the wild-type GCN4 allele and a strain containing a GCN4 allele which was unresponsive to the translational control due to point mutations of the four uORFs.
We observed a biphasic response of Gcn4 protein levels, Gcn4p DNA binding activity and activation ability in the cell upon the signal "amino acid starvation." The first phase started immediately after induction by 3-AT and is due to the translational control depending on the four uORFs. After 3-4 h of starvation, an additional 2-fold increase started which seems to be due to increased transcriptional initiation induced by amino acid starvation.
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EXPERIMENTAL PROCEDURES |
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Plasmids
Construction of the Gcn4p Reporter Plasmid--
pME1112 and
pME1108 were constructed using the yeast integration vector pLI4 (18)
containing the CYC1::lacZ reporter gene (19). pME1112 (GCRE6::lacZ) was used to integrate
a Gcn4p regulated lacZ gene carrying six general control
responsive elements (GCRE) in the CYC1 promoter region into
the genome. The same construct without GCRE was used as control and
named pME1108 (UAS::lacZ). Replacement of the
upstream activation site (UAS) of the
CYC1::lacZ with GCREs was achieved by
first cutting out the XhoI fragment containing the UAS.
Religation of the XhoI site resulted in pME1108. Then, two synthetic oligonucleotides (GAC1, 5'-GATCGGATGACTCATTTTTT-3' and GAC2, 5'-GATCAAAAAATGAGTCATCC-3') were annealed to form an optimal double-stranded GCRE (20). T4 DNA ligase was added to produce
multimeric GCRE DNA probes. The products were then blunt ended with
Klenow and inserted into the blunt ended XhoI site of
pME1108. The resulting plasmid pME112 contains six GCRE sites as
determined by nucleotide sequence analysis (21). Enzymatic manipulation
and cloning of DNA were performed as described by Sambrook et
al. (22).
Construction of a GCN4 Deletion Cassette--
A 2.8-kilobase
EcoRI-SalI fragment containing the
GCN4 gene was isolated from vector p164 (12) and inserted
between the EcoRI and SalI sites of pBluescribe
M13 to get vector pME1085. The complete GCN4
open reading frame was deleted by removing a
BstEII-BfrI fragment. A blunt ended
HpaI-NarI fragment containing the LEU2
gene isolated from vector YEp351 (23) was then inserted between the
blunt ended BstEII and BfrI sites to obtain
pME1105.
Construction of GCN4 and GCN4m Carrying Centromeric Vectors-- A 2.8-kilobase EcoRI-SalI segment containing the GCN4 gene was isolated from vector p164 (12) and inserted between the EcoRI and SalI sites of vector pRS314 (24) to get vector pGCN4 (pME1092). The 640-bp SalI-BstEII fragment of pGCN4 carrying the four short open reading frames in the GCN4 leader region was then replaced by a 640-bp SalI-BstEII fragment of vector p238 containing four mutated (non-functional) open reading frames in the GCN4 leader region (13) to get vector pGCN4m (pME1098).
GCN4::lacZ Fusion Plasmids p180 and p227-- The plasmids were kindly provided by Alan Hinnebusch (National Institute of Child Health and Human Development, Bethesda, MD). p180 is a URA3,RS1,CEN4 plasmid containing a GCN4::lacZ fusion with lacZ fused in-frame at the 55th codon of GCN4 (12). p227 is a derivative of p180 containing substitution mutations in the AUG codons of all four uORFs in the GCN4 mRNA leader (25).
Yeast Strains and Growth Conditions
RH1796 (MATa ade2-101 leu2-3 leu2-112 suc2-9
trp1-
901 ura3-52::GCRE6::lacZ
gcn4::LEU2) and RH1798 (MATa ade2-101 leu2-3 leu2-112 suc2-
9 trp1-
901 ura3-52::
UAS::lacZ
gcn4::LEU2) are derivatives of
S. cerevisiae strain SEY6210 (MATa ade2-101
his3-
200 leu2-3 leu2-112 suc2-
9 trp1-
901 ura3-52;
obtained from Scott Emr, University of California San Diego). Wild-type
strain RH1415 (Mat
ura3-52 leu2) was described earlier
(26).
Strains RH1796 and RH1798 were constructed by first performing a
one-step replacement of the chromosomal GCN4 gene using the ScaI fragment of vector pME1105 harboring the
GCN4 deletion cassette. Then the His phenotype
was rescued by transformation with a linear
AatII-NaeI fragment containing the intact
HIS3 gene from vector pRS303 (24). Finally, the
StuI-linearized plasmids pME1112 and pME1108, respectively, were integrated at the ura3-52 locus. Transformations were
carried out using the lithium-acetate yeast transformation method (27). Correct integration or replacement of all manipulations was confirmed by Southern blot analysis (28).
Yeast cells were cultivated on minimal medium (29). Appropriate
supplements were added in recommended amounts (30). Routinely precultures were grown overnight, diluted, and cultivated for another
12 h to an optical density of approximately 0.5 at 546 nm. Then 40 mM 3-aminotriazole was added to induce histidine
starvation. After the appropriate growth period, cultures were divided
and harvested for parallel measurement of -galactosidase activity and protein or RNA isolation.
Northern Blot Analysis
Total RNA from yeast was isolated following the protocol described by Zitomer and Hall (31). 10 µg of total RNA from each sample was separated on a formaldehyde agarose gel, electroblotted onto a nylon membrane, and hybridized with 32P-labeled probe prepared from a 560-bp polymerase chain reaction-generated GCN4 fragment using the oligolabeling technique described by Feinberg and Vogelstein (32). A 32P-labeled 540-bp ClaI fragment of ACT1 was used as internal standard. Hybridizing signals were quantified using a BAS-1500 PhosphorImaging scanner (Fuji).
Western Blot Analysis and the Production of Anti-Gcn4p Antibodies
Yeast protein crude extracts for Western blot analysis were prepared as described (33). Protein contents were measured using a Bio-Rad protein assay kit according to Bradford (34). 25 µg of protein from crude cell extracts were separated on a 12% SDS-polyacrylamide gel and subsequently analyzed by Western blot analysis using polyclonal antibodies raised against the COOH terminus of Gcn4p. Pure Gcn4p (10 ng) expressed and isolated from Escherichia coli (9, 35) was loaded as a control. Immune complexes were visualized with an alkaline phosphatase ImmunoBlot assay kit (Bio-Rad). Gcn4p signals were quantified using the Molecular Analyst software from Bio-Rad. For calibration, a standard curve derived from measurements of defined amounts of pure Gcn4p was used.
Polyclonal anti-Gcn4p antibodies were raised in a rabbit against a synthetic peptide containing the 60 COOH-terminal amino acids of Gcn4p. The peptide was linked to keyhole limpet hemocyanin with glutaraldehyde and injected subcutaneously after being mixed with an equal volume of incomplete Freud's adjuvant. After 5 weeks, additional injections (six times, once per week) served as boosts.
Gel Retardation Assay
The gel retardation assay using Gcn4 protein expressed in
E. coli was described earlier (36). The DNA probe was a
32P-end-labeled synthetic 49-bp TRP4-UAS1 DNA
fragment (37). The TRP4-UAS1 fragment was obtained by
annealing of two synthetic oligonucleotides identical to the
TRP4 promoter region 273 to
225 containing one GCRE
site. 15 µg of protein extracts were incubated with 10 fmol of
32P-radiolabeled probe, separated on a native 6%
polyacylamide gel, and visualized by autoradiography. UAS1-Gcn4p
signals were quantified using a BAS-1500 PhosphorImaging scanner
(Fuji).
-Galactosidase Assay
-Galactosidase activities of permeabilized yeast cells were
determined by using 4-methylumbelliferyl-
-D-galactoside
as a fluorogenic substrate (38). Yeast cells were cultivated in minimal medium. After addition of 3-AT, specific
-galactosidase activities were assayed. Concentration of product formation during the reaction was determined based on a standard curve with commercial products (4-methylumbelliferone; Fluka, Buchs, Switzerland). Product
concentrations were normalized to the reaction time and the optical
density of the culture. One unit of
-galactosidase activity is
defined as 1 nmol of 4-methylumbelliferone h
1
ml
1 OD546
1. The given
values are means from measurements of at least five independent
cultures. The standard errors of the means were below 25%.
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RESULTS |
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Histidine Starvation by 3-Aminotriazole Leads to 2-Fold Increased
GCN4 mRNA Transcript Levels--
We performed a kinetic analysis
of the steady state amount of GCN4 mRNA after induction
of the general control system by 3-AT. A yeast strain carrying a
deletion of the chromosomal GCN4-locus (RH1796,
gcn4 strain) was transformed with either the wild-type GCN4 allele (pGCN4) or a mutated GCN4 allele
(pGCN4m) on a centromer-based plasmid. The translational
control of mRNA expression of the GCN4 allele of
pGCN4m is abolished by point mutations in the start codons
of the four uORFs (13). Histidine starvation was induced by adding 3-AT to exponentially growing cultures. Samples were taken at different time
points and GCN4 mRNA levels were subsequently analyzed
by Northern blot analysis and quantified. GCN4 expression
from both alleles, wild-type GCN4 as well as
GCN4m, showed identical kinetics after induction
by 3-AT (Fig. 1). A 2-fold increase in
GCN4 mRNA steady state levels was reached after
approximately 2 h and remained constant for at least 6 h.
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Gcn4 Protein Levels Increase in Two Steps after Histidine Starvation-- The changes of Gcn4 protein levels in yeast cells after the switch to histidine starvation conditions were analyzed in Western blot hybridization experiments. Crude protein extracts were prepared from cells cultivated for different time periods after addition of 3-AT to the medium. Gcn4 protein levels were assayed using a polyclonal antiserum raised against the DNA-binding domain of Gcn4p. In a strain harboring the wild-type GCN4 allele, a biphasic increase in Gcn4p after addition of 3-AT was observed (Fig. 3, A and C). Elevation of Gcn4 protein levels started after 20 min and increased by a factor of 3 during the first hour. Afterward, Gcn4 protein levels remained constant for 2 h. Three to four hours after induction an additional 2-fold increase was observed leading to a 6-fold total increase in the amount of Gcn4p after the initial induction by 3-AT.
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Gcn4p DNA Binding Activity Correlates with Changes of Gcn4p Levels-- The increased amount of Gcn4 protein upon amino acid starvation prompted us to analyze whether increased protein levels also correlate with increased functionality. As a first function of the transcriptional activator Gcn4p we analyzed its ability to bind to its specific DNA-binding site. Therefore, we established a gel retardation assay using crude protein extracts. Yeast cells were grown 8 h on minimal medium in the presence or absence of 3-AT. Crude extracts including Gcn4p expressed from pGCN4 were tested for specific binding to a synthetic DNA fragment that contains a Gcn4p responsive element originally derived from the TRP4 promoter (37). Induction of the general control system by 3-AT resulted in an increased intensity of the retarded band, suggesting an increased DNA binding activity in yeast crude cell extracts. The increased DNA binding activity correlated with the increased amount of Gcn4 protein in the cell as described above, suggesting that the affinity of the protein to its DNA was unaffected and only the amount of DNA-binding protein increased (Fig. 4A).
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A General Control Regulated Reporter Gene Responds in Two Equivalent Phases to the Amino Acid Starvation Signal-- We wanted to analyze whether the two-step increase of the Gcn4 protein after 3-AT induced histidine starvation is also reflected in the induction of a general control regulated target gene. To make sure that even small changes in the activation of the Gcn4p regulated gene can be detected, we constructed an artificial general control reporter gene based on a CYC1::lacZ fusion construct (19). The promotor region of the CYC1::lacZ gene was replaced by six Gcn4p responsive elements resulting in a highly sensitive general control regulated target gene. This construct was stably integrated into yeast strain RH1796 at its ura3-52 locus. The consequences of the observed biphasic increase of the Gcn4p levels were monitored by the transactivation ability of Gcn4 protein using this Gcn4p regulated CYC1::lacZ-reporter gene (Fig. 5A).
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DISCUSSION |
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We examined the time course of the Gcn4 protein elevation of yeast cells after switch to amino acid starvation cultivation medium induced by 3-aminotriazole. GCN4 mRNA is already present in the cell under nonstarvation conditions, but only under starvation conditions does efficient translation occur (39). In this report, we find that amino acid starvation induced by 3-AT results in a biphasic response of the cell. A first step of increased GCN4 expression is caused by translational control and depends on the four uORFs of the GCN4 leader region. As a consequence, more Gcn4 protein is produced within 20 min after histidine starvation. In addition, GCN4 mRNA levels increase and subsequently a second elevation phase of Gcn4 protein levels can be observed 4-6 h after induction. Accordingly, expression of a Gcn4p-dependent CYC1::lacZ reporter gene also responds in two phases. Cells with a nonfunctional translational control of GCN4 show only the second phase of response under the same conditions.
The elevation of GCN4 transcript levels after induction of the general control system raises the question of whether this increase is due to changes in stability of the GCN4 mRNA or whether it is due to an increased transcription of GCN4. The GCN4::lacZ fusion constructs suggest that histidine starvation rather affects transcription initiation of GCN4 than the stability of the mRNA. It has been shown that the gene expression of the Gcn4p homologues cpcAp of Aspergillus niger and CPC1 protein of Neurospora crassa are regulated at a transcriptional level (40, 41). However, transcriptional regulators of GCN4 are not known yet. In S. cerevisiae, premature translation termination promotes rapid degradation of numerous mRNAs (42). It was speculated that the four uORFs preceding the GCN4 coding region at which translational initiation and reinitiation events occur would function in a manner analogous to nonsense codons, promoting rapid degradation of the mRNA (43). However, previous studies demonstrated that the GCN4 transcript is not degraded by the nonsense-mediated mRNA decay pathway and reported a half-life of 16-18 min of GCN4 mRNA (44). Whereas it cannot be excluded, that there is regulated GCN4 mRNA degradation in yeast, we do not think that this is important under the conditions we have tested here.
The DNA binding activity and the ability to activate transcription of
target genes both correlate with the amount of Gcn4 protein in the
cell. However, there are quantitative differences between the ratio of
Gcn4p levels and of -galactosidase activities of corresponding
GCN4::lacZ fusion constructs during the
amino acid starvation kinetic experiments. This is presumably due to differences in protein stability compared with the native Gcn4 protein
and the chimeric protein encoded by the
GCN4::lacZ fusion constructs. The
half-life of
-galactosidase expressed in S. cerevisiae is
over 20 h (45). It is also known that the half-lives of chimeric
-galactosidase fusion proteins can possibly range between 3 min and
over 20 h, depending on the nature of the amino acids at the amino
terminus of the protein (45). The used
GCN4::lacZ fusion constructs include
only 55 amino acids of the amino terminus of Gcn4p excluding the
reported instability region of Gcn4p (17). Thus, it is likely that the
chimeric protein encoded by the
GCN4::lacZ fusion lacking the
translational control accumulates also under nonstarvation conditions.
In contrast, Gcn4 protein under nonstarvation conditions is
highly unstable with a reported half-life of 5 min (17). Therefore,
stabilization of Gcn4 protein plays an additional role under
certain circumstances (17).
The observed results also raise the question of how a 6-fold increase in Gcn4p levels in the cell is sufficient to elevate expression of at least 40 genes 2-5-fold. One possibility would be an improved DNA binding ability of Gcn4p under starvation conditions as found for the human oncoprotein c-Jun that displays a regulated DNA binding activity (46). It was shown that the DNA-binding domains of c-Jun and Gcn4p are functionally homologous (47). Phosphorylation of distinct sites near the DNA-binding domain inhibits DNA-binding and dephosphorylation correlates with increased transactivation activity of c-Jun (46). In contrast, Gcn4 protein levels and its ability to bind DNA in vitro correlate in time and value, suggesting that there is no such post-translational regulation of Gcn4p. Therefore, we conclude that the DNA binding ability is not affected under the tested conditions. Another possibility could be a regulated nuclear localization of Gcn4p. Such a regulation is shown for the yAP-1 protein (48). The corresponding YAP1 gene encodes another member of the leucine zipper family and recognizes a Gcn4p DNA-binding sequence motif (49). Upon imposition of oxidative stress, a small increase in the DNA binding capacity of yAP-1 protein occurs (48). However, the major change is at the level of nuclear localization; upon induction the yAP-1 protein relocalizes from the cytoplasm to the nucleus (48). It remains to be elucidated whether a similar transport regulation mechanism will be found for Gcn4p.
In summary, our results suggest the existence of an additional mechanism to control Gcn4p levels upon amino acid limitation in yeast. So far, translational control via the uORFs in GCN4 has been found to be the major mechanism to regulate GCN4 expression. This report shows that upon prolonged amino acid starvation Gcn4p levels can be increased by a mechanism independent of the uORFs. Whether this mechanism includes elements of the known general control system or consists of as yet novel factors remains to be elucidated in the future.
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ACKNOWLEDGEMENTS |
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We are grateful to Eric Kübler and Katrin Düvel for critical reading of the manuscript and all other members of the group for helpful discussions. We especially thank Alan Hinnebusch for kindly providing plasmids.
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FOOTNOTES |
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* This work was supported by Deutsche Forschungsgemeinschaft, Fonds der Chemischen Industrie, and Volkswagen-Stiftung.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Institut für
Mikrobiologie und Genetik, Abteilung für Molekulare
Mikrobiologie, Grisebachstr. 8, D-37077 Göttingen. Germany.
Tel.: 49-0-551-39-3771; Fax: 49-0-551-39-3820; E-mail:
gbraus{at}gwdg.de.
1 The abbreviations used are: 3-AT, 3-aminotriazole; eIF-2, eukaryotic initiation factor 2; eIF-2B, guanine nucleotide exchange factor of eIF-2; GCRE, general control responsive element; tRNAiMet, initiator tRNA charged with methionine; UAS, upstream activation site; uORF, small upstream open reading frame; bp, base pair(s).
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REFERENCES |
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