Cell, Vol. 70, 647-657,

August 21, 1992, Copyright

0 1992 by Cell Press

Autoregulation of the Yeast Lysyl-tRNA Synthetase Gene GCDSIKRSI by Translational and Transcriptional Control Mechanisms Stefan Lanker,’ Janet L. Bushman,t Alan G. Hinnebusch,t Hans Trachsel,’ and Peter P. Mueller’ ‘Institute of Biochemistry and Molecular Biology University of Berne 3000 Berne 9 Switzerland fsection on Molecular Genetics of Lower Eukaryotes National Institute of Child Health and Human Development National Institutes of Health Bethesda, Maryland 20892

Summary We cloned the GCDS gene of S. cerevisiae and found it to be identical to KRS7, which encodes lysyl-tRNA synthetase (LysRS). The mutatlon g&S-7 changes a conserved residue in the putative lysine-binding domain of LysRS. This leads to a defect in lysine binding and, consequently, to reduced charging of tRNALyg. Mutant gcd5-7 cells compensate for the defect in LysRS by increasing GCN4 expression at the translational level. GCN4 protein in turn stimulates transcription of GCD5, leading to Increased LysRS activity. We propose an autoregulatory model in which uncharged tRNALp stimulates the protein kinase GCNS, a translational activator of GCfU4, and thereby increases transcription of GCDS and other genes regulated by GCN4. Introduction Starvation of the yeast Saccharomyces cerevisiae for a single amino acid leads to increased synthesis of GCN4, the transcriptional activator of a number of unlinked genes encoding enzymes involved in amino acid biosynthesis (general amino acid control). GCN4 expression is controlled at the translational level by four short upstream open reading frames (uORFs) in the mFlNA leader that reduce translation when amino acids are abundant but allow increased translation of GCN4 in response to amino acid starvation (for reviews see Hinnebusch, 1988, 1990; Hinnebusch and Klausner, 1991). A recently proposed model (Abastado et al., 1991) suggests that under nonstarvation conditions, ribosomes scanning from the mRNA cap initiate at the first uORF encountered (uORF1) and following termination resume scanning and reinitiate translation at one of the downstream uORFs (2, 3, or 4). After translating one of these uORFs, ribosomes are released from the mRNA; therefore, only a small fraction of the ribosomes initiate translation of the GCN4 ORF. Under conditions of amino acid starvation, ribosomes that resume scanning after translating uORF1 become competent for reinitiation more slowly than those under nonstarvation conditions. Therefore, a significant fraction of these ribosomes bypass uORFs

2-4 and reinitiate at the GCN4 ORF instead. It was recently demonstrated (Dever et al., 1992) that the protein kinase GCNP, a positive regulator of GCN4 expression (Wek et al., 1989) phosphorylates translation initation factor 2 (elF-2) on its a subunit under amino acid-starvation conditions. Phosphorylation of mammalian elF-Pa has long been known to inhibit protein synthesis at the initiation step (reviewed in Moldave, 1985; London et al., 1987). Dever et al. proposed that, because of reduced amounts of active elF-2, reinitiation of translation is less efficient under starvation conditions, allowing more ribosomes to scan past uORFs 2-4 and initiate at the GCN4 start codon. This model is supported by the fact that mutations in serine 51 of elF9a, the site of phosphorylation on elF9a in mammalian cells (Colthurst et al., 1987) impair translational derepression of GCN4 expression (Dever i : al., 1992). It is thought that accumulation of uncharged tRNA in amino acid-starved cells stimulates GCNP to phosphorylate elF-2a because the i/.sl-7 mutation affecting isoleucyltRNA synthetase leads to derepression of the general control system in amino acid-complete medium (Delforge et al., 1975). The GCNP protein contains a large domain related to histidyl-tRNA synthetase (HisRS) adjacent to its protein kinase moiety, and point mutations in this HisRSlike region impair the derepression of GCN4 translation in response to amino acid starvation (Wek et al., 1989). These observations led to the suggestion that binding of uncharged tRNA to the HisRS-like region increases the activity of the protein kinase domain of GCN2. Presumably, the HisRS-related region of GCNP has diverged sufficiently from authentic HisRS so that it would bind other uncharged tRNAs besides tRNAHiJ (Wek et al., 1989). Mutants lacking GCN2 kinase function cannot efficiently translate GCN4 mRNA under amino acid-starvation conditions (Gcn- [general control nonderepressible] phenotype). By contrast, mutations in multiple GCD genes lead to high level translation of GCN4 mRNA in the absence of amino acid starvation (Gcd- [general control constitutively derepressed] phenotype), the same phenotype associated with reduced elF-2 activity. Most gcd mutations are pleiotropic, leading to slow growth or temperature-sensitive growth under nonstarvation conditions besides derepressing GCN4 expression (Hinnebusch, 1990). This dual phenotype suggests that GCD proteins have an essential function in addition to the regulation of GCN4 expression. Recent evidence indicates that GCDl (Tzamarias et al., 1989; Cigan et al., 1991) and GCD2 (Foiani et al, 1991) are required for general translation initiation, perhaps as components of the eukaryotic initiation factor 28 (elF-PB), which catalyzes GDP-GTP exchange in elF-2 and thereby stimulates new rounds of translation initiation. elF-2 must be complexed with GTP to bring initiator tRNA to the small ribosomal subunit and form the 43s preinitiation complex that binds to mRNA and scans to the AUG start codon (reviewed in Moldave, 1985). Phosphorylation of elF-Pa in mammalian cells inhibits the recycling of elF-2.GDP to elF-2’GTP catalyzed by elF-2B (Hershey, 1991). Thus, a

gcd mutation that reduces elF-PB function would mimic the inhibitory effect of ptiosphorylation on elF-2 activity. In this report we characterize the defect associated with the gcd5-7 allele. This mutation, along with several others, including gcd2-7, was isolated in a selection for strains resistant to amino acid analogs (Niederberger et al., 1988). Unlike other gcd mutations analyzed, gcd5-7 was found to be lethal in combination with gcnl, gcn2, gcn3, or gcn4 mutations. This synthetic lethality suggested that GCD5 may affect the expression of enzymes subject to the general control response by a mechanism distinct from the proposed functions of GCDl and GCD2 in regulating elF-2 activity. Therefore, we initiated a molecular analysis of GCDS to characterize its biochemical function and its role in modulating GCN4 expression. Results Clonlng of the GCDI Gene The gcd5-7 mutation was isolated on the basis of causing increased resistance to the tryptophan analog Bfluorotryptophan (5.Ff), a phenotype indicative of constitutive derepression of enzymes subject to general amino acid control. The mutation also leads to slow growth (Slg-) on nutrient-rich or minimal medium (Niederberger et al., 1988). The GCDS gene was isolated from a yeast genomic library on plasmid pJBl2 by its ability to complement the Slg- and 5-FT resistance (5.FT9 phenotypes of the gcd5-7 mutation (see Experimental Procedures). We localized the GCD5 gene on the genomic insert of pJB12 by testing various subclones for the ability to complement the slowgrowth phenotype of gcd5-7. RNA blot hybridization analysis of total yeast RNA using the subcloned fragments as probes revealed a transcript of -2.0 kb encoded by the central portion of the insert in pJBl2 that coincided with the sequence required for complementation (data not shown). We therefore chose to sequence the ends of several of the subcloned fragments derived from pJB12. A data base search with the resulting seven sequences, each 200300 bp in length, revealed 100% identity with the ORF or flanking sequences of the KRS7 gene, encoding the yeast lysyl-tRNA synthetase (LysRS; Mirande and Wailer, 1988). The identity of GCDS and KRS7 was further substantiated by determining that the restriction map of the entire 5 kb insert in pJB12 was identical to that of the previously published 5.2 kb sequence of the KRS7 region. The gcd5-7 Mutation Leads to Reduced Lysyl-tRNA-Charging Activity Having identified GCDS as KRS7, we wished to determine whether the gcd5-7 mutation affects LysRS activity. To address this question, we performed in vitro Iysyl-tRNAcharging assays. Extracts prepared from wild-type and gcd5-7 strains were assayed under conditions of limiting extract for incorporation of radioactively labeled lysine into a trichloroacetic acid (TCA)-precipitable species. The gcd5-7 mutant extract showed a P-fold reduction in the activity of [3H]lysine incorporation into tRNA compared with wild type (Figure 1A). We also examined the level

A 3 H-lysine I

B ’%

I

amino

C acids

WI.

activity

I

Figure 1. LysRS Activity in gcd6-I Mutant and Wild-Type Extracts Yeast extractsof strainsT66A(gc&l)and GRFlB(wt) were prepared, and tRNA synthetase activities were measured as described in Experimental Procedures. Each reaction mixture (50 ul) contained 3-4 ug of protein and 1 uCi of SH-labeledlysine (A) or 1 uCi of 14C-labeledtotal amino acids(B). Specific activities were calculated as TCA-precipitable counts per minute (charged tRNA) formed per minute per milligram protein. (A) Lysyl-tRNA-charging activity assayed using [3H]lysine; the value of the wild-type strain was set to 100%. (B) Total tRNA-charging activity assayed using a mixture of “C-labeled amino acids; the value of the gcd5 mutant strain was set to 100%. (C) Relative lysyl-tRNA-charging activity, calculated as Iysyl-tRNAcharging activity divided by total tRNA charging activity.

of total aminoacyl-tRNA synthetase activities by adding a radioactively labeled mixture of all amino acids to extracts of wild-type and mutant strains (Figure 1B). Consistent with its Gcd- phenotype (see Discussion for further details), the overall synthetase activity in the gcd5-7 mutant was 2.5.fold higher than in the wild-type strain. Consequently, the amount of LysRS activity relative to total tRNA-aminoacylation activity in the gcd5-7 mutant was only approximately 20% of that seen in the wild-type strain (Figure 1C). When the mutant was transformed with plasmid pJB12 containing GCDS, or with the vector YCp50 alone, and assayed for LysRS activity, we observed 2.5. to 3.fold greater activity in extracts from the pJBl2 transformants compared with the YCp50 transformants (data not shown). Thus, the gcd5-7 mutation is responsible for the reduced tRNALYs-charging activity observed in the mutant extract. The gcd5-7 Mutation Lowers the Affinity of LysRS for Lyslne Reduced charging activity of the gcd5-l-encoded LysRS could result from impaired binding of any one of the three substrates: ATP, lysine, or tRNALp. We examined this possibility by conducting in vitro lysyl-tRNAcharging assays with varying concentrations of each substrate. The results show that the mutant synthetase had fairly similar affinities for ATP (Figure 2A) and tRNA (Figure 28) showing a 2.5. fold higher KM for ATP and a 2-fold lower KM for tRNA compared with wild type. However, the affinity of the mutant synthetase for lysine was more dramatically altered (Figure 2C), with a KM S-fold higher than wild type. These results suggest that the major defect of the gcd5-7 mutant LysRS is in binding lysine.

t&oregulation

of GCDS by the General Control System

Figure 2. Substrate Saturation Curves for LysRS Activity and Effect of Lysine on the Growth Rate Experiments were performed as described in Figure 1. In each experiment, the highest values of lysyl-tRNA-charging activity for wild-type (closed triangles) andgcd67 (open circles) mutant strains were set to 100%. (A) ATP dependence. (6) Total yeast tRNA dependence. (C) Lysine dependence of lysyl-tRNA formation. A&: difference of KMvalues for wild-type and mutant strains. (D) and(E) The gcd57 mutant strain T66A was transformed with pJB12 bearing GCD5 (wt) or vector alone (gcd57). Two transformants of each type were grown for 4 days at 30% on SD plates containing either arginine (2 mglml [D]) or lysine (3 mglml [El).

Lysine [pM]

Excess Lysine Can Reduce the Slow-Growth Phenotype of g&S-l Because themutantsynthetaseshowedadecreasedaffinity for lysine, we addressed the possibility that addition of exogenous lysine would diminish the slow-growth phenotype of the gcdfj-1 mutant. The gccl5-1 strain T88A was transformed with plasmid pJBl2 containing GCDS or with vector alone, and the resulting transformants were grown on minimal agar medium supplemented with the necessary amino acids and either excess lysine (3 mglml; Figure 2E) or, as a control, excess arginine (2 mg/ml; Figure 2D). The lysine supplement appeared toshorten thegeneration time of agcd57 mutant strain, increasing the size of colonies formed from single cells after 4 days of incubation relative to that seen on plates supplemented with arginine. By contrast, GCD5 transformants formed equally large colonies on both media. In liquid minimal medium, the generation time of the GCD5 transformant was slightly greater in the presence of 20 t&l lysine (200 min without lysine, 220 min with lysine), whereas the generation time of the gcd5-1 transformant was decreased by 35% in the pres-

ence of 20 HIM lysine (500 min without lysine, 320 min with lysine). These results are consistent with a lysine-binding defect for the LysRS encoded by gcd5-1. The gcd5-7 Allele Has a Single Point Mutation In the Putative Lysine-Binding Domain of LysRS To investigate the molecular basis of the lysine binding defect of the mutant enzyme, we decided to sequence the gcd5-7 allele. We used the polymerase chain reaction (PCR) with two primers immediately 5’and 3’to the GCDS ORF to amplify a 1.94 kb fragment containing the genomic gcd5-7 allele. A single point mutation was detected in the entire sequence of the mutant 1.94 kb fragment (Figure 3A): a G-+T transversion at position 1483 that changes arginine 488 to leucine. This mutation lies in the proposed amino acid-binding domain of LysRS (Cusacket al., 1991) and affects a residue conserved between Escherichia coli and S. cerevisiae (Figure 36). This finding suggests that the gcd5-7 mutation directly impairs binding of lysine by altering the structure of the lysine-binding domain of LysRS.

Cdl 650

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484 EC 409

Glu Ile Thr dsp

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1486 AAG QAA MT ‘E-l’ AAT CCC TAC ACT QA& sc 4% LysQluIle~s~dla~rThrOluLsuAwllsp EC 421 drg5luIleGlyAmGlyPheSerGluLeuAnndsP

TlG

A&C QNl’

Figure 3. Amplification and Sequencing of the Mutant g&5-7 Allele Genomic DNA segments from GRF16 (wt) and T66A (gcd5-1) were amplified with the PCR as described in Experimental Procedures. (A) Autoradiogram showing wild-type (wt) and mutant (gcd57) sequences in the vicinity of the single point mutation GIW+T detected in the g&5-7 allele. (8) Alignment of a portion of the sequences of S. cerevisiae (SC) and E. coli (EC) LysRSs in the putative lysine-binding domain. Amino acids are in italics, and conserved residues are in bold letters.

gcdbl Leads to Derepression of GCN4 Expression The gcd5-1 mutation leads to derepression of the TRP3 gene, expression of which is regulated by the general amino acid control (Niederberger et al., 1988). Because GCN4 is the proximal transcriptional activator in this system, we investigated the effect of gcd5-1 on GCN4 expression. A plasmid containing a GCN4-/acZ fusion (Hinnebusch, 1984) was introduced into gcd5-1 mutant and wild-type strains, and the transformants were characterized for the levels of 9-galactosidase activity expressed under different conditions. In minimal medium (Figure 4A, unsupplemented), GCN4-lacZ expression was more than 1Bfold higher in the gcd5-1 strain than in the wild-type strain. Upon starvation of the strains for histidine by addition of 3-aminotriazole, an inhibitor of the HIS3 enzyme (Figure 4A, histidine starved), GCN4-/acZexpression was derepressed approximately g-fold in the wild-type strain, whereas expression in the gcd5-1 mutant was slightly reduced to approximately wild-type derepressed levels. The

addition of excess lysine to minimal medium, shown above to increase the growth rate of the gcd5-1 strain, reduced GCN4-/acZ expression in the gcd5- 1 mutant by a factor of 8 (Figure 4A, lysine supplemented), whereas lysine addition had little or no effect in the wild-type strain. These results show that the gcd5-1 mutation leads to an increase in GCN4 expression that can be overcome by addition of excess lysine. The simplest explanation for these findings is that, in the absence of exogenous lysine, the gcd5-1 mutation leads to a deficiency in charging of tRNALys that signals the derepression of GCN4 . Toverify that the derepression of GCN4 expression seen in the gcd5-1 mutant is mediated at the translational level by the uORFs in the GCN4 mRNA leader (Mueller and Hinnebusch, 1988), we introduced a GCN4C-/acZ fusion lacking all four uORFs into gcd5-1 mutant and wild-type strains and analyzed the resulting transformants for expression of b-galactosidase activity. In both strains, GCN4”-/acZ expression was very high (- 2000 U) and showed little or no additional derepression upon histidine starvation (data not shown). This result was expected for the GCDS strain because the uORFs are required for repression of GCN4 translation under nonstarvation conditions and for its derepression in response to histidine starvation (Mueller and Hinnebusch, 1988). The fact that gcd5-1 increases expression of P-galactosidase activity 12-fold from the construct containing the uORFs and only 1 X-fold from the construct lacking uORFs is a strong indication that the mutation derepresses GCN4 at the translational level (Mueller and Hinnebusch, 1988). Further support for this conclusion was provided by measuring the steady-state levels of GCN4 and GCN4-/acZ mRNAs in the gcd5-1 mutant and wild-type strains by RNA blot hybridization analysis. The results showed that the levels of these mRNAs differed between the two strains by less than a factor of 2 (data not shown). GCDS-IacZ Expression Is Derepressed in gcdl-f Strains Since GCDS has a consensus GCNCbinding site (TGACTC box) in its 5’noncoding sequence (Mirande and Wailer, 1988) we reasoned that the elevated GCN4 expression seen in the gcd5-1 mutant might lead to an increase in GCDS transcription and KRS enzyme levels. To analyze the effects of gcd5-1 on GCDB expression, we constructed a GCDS-/acZ fusion and introduced it into gcd5-1 mutant and wild-type strains. RNA blot hybridization analysis showed that the steady-state level of GCDS mRNA was increased substantially (4.4.fold) in thegcd5-1 mutant relative to wild type when both strains were grown on minimal medium (compare lanes 1 and 3, Figure 48). A smaller, but significant (Bfold), increase in GCDS-/acZ mRNA was also observed in the mutant (Figure 48, lane 3). Addition of excess lysine reduced the GCDB and GCD5-/acZ transcript levels in the gcd5-1 mutant strain (lane 4), whereas the GCD5 mRNA level in the wild-type strain was affected very little by lysine (lane 2). Measurements of GCD5-lacZ fusion enzyme activity in the two strains (Figure 4C) gave results very similar to those just discussed for the GCDS transcript (Figure 4S),

Autoregulation 651

of GCD5 by the General Control System

B lysine:

Wt -

+

gcd5 1 -

+

Figure 4. Analysis of GCN4 Expression in gcd5-7 Mutant and Wild-Type Strains and Regulation of GCDB Expression by GCN4

GCiV4-/ecZ (A) or GCDWacZ (B, C, and D) fusion constructs were introduced into wildtype (wt) and gcdd7 mutant strains T15OA and 7.45.3_i :i -GCDdlacZ T66A, respectively (A, B, and C), a gcn4 mutant ([D], gcn4), or a strain with high-level constitutive expression of GCAf4 ([D], GCN43. (A) Steady-state levels of p-galactosidase activity expressed from GCN4-/acZ in gcd5-l or wild-type strains were determined from transformants grown under normal conditions (ungcdS-1 supplemented), under histidine-starvation conditions (histidine starved), or in the presence of excess exogenous lysine (lysine supplemented). (B) Steady-state levels of GCDS and GCD5lacZ mRNAs. Strains were grown in minimal medium containing only the required nutrients (lanes 1 and 3) or in medium supplemented with excess lysine (3 mg/ml; lanes 2 and 4). Total RNA from g&i-I mutant (lanes 3 and 4) and wild-type strains (lanes 1 and 2) was extracted and separated by electrophoresis on a 1% formaldehyde-agarose gel, blotted to a nitrocellulose membrane, and hybridized to a GCD5-lacZ-specific probe as described in Experimental Procedures. The intensities of individual bands were quantified by densitometric analysis. Positions corresponding in size to GCN4” gcn4 GCD5-/acZ mRNA or GCD5 mRNA are shown on the right. Positions of RNA size markers are given on the left. (C)Steady-state levels of 6-galactosidase activity expressed from GCDb-/acZ in gcd5-7 mutant or wild-type strains grown in minimal medium (unsupplemented) or minimal medium supplemented with excess lysine (lysine supplemented). (D) Steady-state levels of f3-galactosidase activity expressed from GCDB-/ecZ in gcn4 and GCN4C strains grown with excess lysine (lysine), excess argine (arginine), or excess lysine and arginine (both). Enzyme activities are expressed in nanomoles of ortho-nitrophenyl+D-galactopyranoside cleaved per minute per milligram of protein. Each value is the mean of three or more independent measurements (less than 25% standard error).

showing 3.9-fold higher expression in the gcd5-7 mutant versus the wild-type strain in minimal medium and a e-fold repression of enzyme activity in the gcd5-7 mutant upon addition of exogenous lysine. By contrast, in the wild-type strain, GCDSlacZ enzyme activity was nearly constant in the presence or absence of lysine (Figure 4C). It is not understood why the level of GCDS-/acZ mRNA is affected less by the gcd5-7 mutation than wild-type GCDB mRNA. Moreover, the GCD5-IacZ enzyme activity is increased to a greater extent than GCDS-IacZ mRNA by the gcd5-1 mutation. This may indicate that GCD5 expression is stimulated in part at a posttranscriptional step in response to decreased charging of tRNALys. In any case, it is clear that theauthenticGCD5 transcript issubstantiallyderepressed in the gcci5-7 mutant. gcd5.1 gcn Double Mutants Can Be Rescued by Overproducing GCN4 The g&5-7 mutation was previously found to be lethal in combination with gcnl, gcn2, gcn3, or gcn4 mutations (Niederberger et al., 1966) each of which impairs derepression of enzymes subject to the general control (Hinnebusch, 1990). The fact that GCD5 encodes LysRS and that this gene isessential (Martinez et al., 1991), combined

with the observation that gcd5-7 leads to increased transcription of GCDS, suggests a model to explain the inviability of gcd5-7 gcn double mutants. We propose that derepression of the gcd5-7 allele is necessary for viability and that this derepression is dependent on the elevated levels of GCN4 protein that occur under starvation conditions and in response to mutations affecting aminoacyl-tRNA synthetases such as gcd5-7. The experiment shown in Figure 4D confirms one of the tenets of our model, that GCD5 expression is regulated by GCN4. We compared expression of the GCDS-/acZ fusion in strains lacking GCN4 function (gcn4) or containing the GCIV~~ allele that expresses high levels of GCN4 protein constitutively owing to the absence of all four uORFs in the GCN4 mRNA leader (Mueller and Hinnebusch, 1966). The results show that GCDS-/acZ expression is substantially higher in the GCJV~~strain compared with the gcn4 mutant, whether cells are grown in a medium containing excess lysine, excess arginine (which leads indirectly to lysine limitation [Delforge et al., 19751) or both amino acids. When we measured tRNALyS-charging activity in extractsof the same gcn4 and GCN4C strains, a 3-fold higher level of activity was observed in the GCN4C strain than in the gcn4 mutant (data not shown), consistent with the results presented in

Figure 4D. These data indicate that GCN4 is a positive regulator of GCD5 transcription. A more incisive test of our hypothesis was conducted by determining whether expression of GCN4 from a constitutive allele can suppress the lethality of gcn gc&-7 double mutants since constitutive GCN4C alleles express GCN4 independent of GCNl, GCN2, and GCN3 (Thireos et al., 1984; Hinnebusch, 1985; Muellerand Hinnebusch, 1988). To test this, we used genetic crosses to construct gcn2 g&5-7 and gcn3 g&5-7 mutants bearing a low copy number plasmid from which GCN4 was expressed at high constitutive levels (see Experimental Procedures). When grown for many generations on nonselective medium, none of the gcn2 gcd5-7 or gcn3 gcd5-7 cells lost the plasmid, indicating that GCN4 overexpression from the allele on the plasmid is necessary for survival of these double mutants. These results strongly support the idea thatgcd5-7 gcn double mutants are lethal because GCN4mediated derepression of GCDS transcription is required for expression of gcd5-1 mutant enzyme activity at a level sufficient for life. Discussion The Amino Acid-Binding Domain of Class 2 tRNA Synthetases Comparisons of the sequences for all 20 aminoacyl-tRNA synthetases from E. coli and several from S. cerevisiae, combined with the high resolution crystal structures of a few of these enzymes, recently allowed the establishment of two distinct classes, to which each synthetase can be uniquely assigned (reviewed in Burbaum and Schimmel, 1991). LysRS belongs to class 2, which is characterized by the presence of three degenerate sequence motifs, called motifs 1, 2, and 3 (reviewed in Cusack et al., 1991). In the threedimensional structures of E. coli seryl-tRNA synthetase (Cusack et al., 1990) and S. cerevisiae aspartyl-tRNA synthetase (Ruff et al., 1991), motif 1 is involved in dimerization, whereas motif 2, motif 3, and the two 8 strands A3 and A4 form the core of an 8-stranded antiparallel 8 sheet that constitutes the active site (Cusack et al., 1991). From the structure of the S. cerevisiae aspartyl-tRNA synthetase-tRNA&P complex (Ruff et al., 1991) it can be seen that motif 2 interacts with the acceptor stem of the tRNA, and there is evidence that motif 3 is involved in binding both ATP and the amino acid (Cusack et al., 1991; Anselme and HattIein, 1991; Kast and Hennecke, 1991). It was proposed that 8 strands A3 and A4 also play a role in amino acid binding because their sequences appear to be uniquely conserved among synthetases specific for the same amino acid in different organisms and because the conserved residues protrude into the active site cleft (Cusack et al., 1991). Our characterization of the gcd5-7 mutation supports a role in amino acid binding for 8 strands A3 and A4 in the active site of class 2 synthetases. We have shown that gcd5-7 is a partially functional allele of the essential gene KRS7 (Martinez et al., 1991) coding for the cytoplasmic LysRS of yeast (Mirande and Waller, 1988). The gcd5-7 allele contains a single point mutation that substitutes the

arginine at position 488 with leucine. Since arginine 488 is one of the conserved residues of 8 strand A3 within the proposed lysine-binding domain of LysRS, one would expect to observe a substantially altered interaction with lysine for the gcd5-l-encoded enzyme. Consistent with this expectation, the Khnvalue for lysine is more than O-fold higher for the gcd5-7 mutant enzyme compared with wild type (Figure 2C). The fact that addition of lysine reduces the slow-growth phenotype of gcd5-7 (Figure 2E) and leads to a reduction in the expression of both GCN4 and GCDS (Figure 4) provides in vivo evidence that the gcd5-7encoded enzyme has reduced affinity for lysine. Taken together, our data support the prediction that 8 strands A3 and A4 in class 2 synthetases form part of the amino acidbinding domain (Cusack et al., 1991). However, it is also possible that the leucine 488 substitution in gcd5-7 in the vicinity of the A3-A4 8 strands alters the function of another structural element that functions in amino acid binding, such as motif 3. An Autoregulatory Model for GCDB Expression, Involving Translational Regulation of GCN4 and Transcrlptional Activation of GCD5 Beginning with the model for GCN4 translational control summarized in the Introduction and the fact that a sequence closely related to the GCNGbinding site is present upstream of GCD5 (5’-ATGACTCTT-3’; Mirande and Waller, 1988) we propose the following autoregulatory model for GCDS expression to account for our results (Figure 5). First, under conditions of insufficient charging of tRNALys by LysRS, GCNP kinase is activated through its HisRSrelated domain and phosphorylates the a subunit of elF-2. This could arise from limiting amounts of lysine or from reduced LysRS activity. Second, phosphorylation of elF2a leads to increased GCN4 protein synthesis (Dever et al., 1992). Third, GCN4 binds upstream of GCD5and stimulates GCD5 transcription. The additional LysRS thus produced increases the level of charged tRNALfl. Consequently, GCNP kinase is no longer activated, phosphorylation of elF-2 returns to basal levels (presumably as the result of dephosphorylation by a protein phosphatase), and translation of GCN4 is once again repressed. The reduced charging activity of the LysRS encoded by gcdli-7 should increase the level of uncharged tRNALw relative to wild-type strains. According to our model, this should stimulate elF-Pa phosphorylation by GCN2, explaining the 1 e-fold derepression of GCN4expression (Figure 4) observed in the gcd5-7 mutant in the absence of an exogenous inhibitor of amino acid biosynthesis. As expected, we found that addition of excess lysine, which should reduce the amount of uncharged tRNAL@, decreased the extent of GCN4 derepression. The increase in GCN4 protein levels that occurs in the gcd5-7 mutant should activate transcription of GCDd In accord with this prediction, the gcd5-7 transcript and GCDB-IacZ fusion protein are expressed at 4-fold higher levels in the mutant strain compared with wild type (Figure 4). This transcriptional activation of gcd5-7 is GCN4 dependent because constitutive high levels of GCN4 protein provided by the GCN4C allele lead to derepressed GCDS-lacZ expression

Autoregulation of GCDS by the General Control System 653

Phosphorylation of elF-2a

5 -1GACTC

dition, gcn2 mutants are sensitive to analogs of various other amino acids (Wolfner et al., 1975). Taken together, these observationssupport the idea that the HisRS-related domain of GCNP interacts with multiple tRNA species in addition to tRNA”‘* (Wek et al., 1989). According to our model, genes encoding aminoacyltRNA synthetases such as GCDS can be autoregulated by making their transcription dependent on GCN4 protein and by signaling translational derepression of GCN4 through the cognate uncharged tRNAs when aminoacylation activity is reduced. Our finding that total tRNAcharging activity was elevated in the gcd5-7 mutant (Figure 28) may be an indication that many different aminoacyl-tRNA synthetase genes are subject to GCN4 control, as shown for isoleucyl-tRNA synthetase (Meussdoerffer and Fink, 1983) and thus autoregulate their expression in the same fashion described here for LysRS. Experlmental Procedures

Figure 5. Autoregulatory Model of GCD5 Expression, Involving Translational Regulation of GCN4 and Transcriptional Activation of GCD5 Uncharged tANA” accumulation is sensed by the HisRS-like domain of the GCN2 protein kinase, leading to activation of the kinase domain (PK). Activated GCNP phosphory(ates elF-2a, and phosphorylated elF-2a causes derepression of GCN4 translation (Dever et al., 1992). GCN4 protein is a transcriptional activator that binds to the TGACTC DNA sequence present upstream of GCDS. This stimulates transcription of GCD5, encoding LysRS. The increased amount of LysRS activity leads to a higher level of lysyl-tRNAL* and a concomitant decrease in uncharged tRNAL*. This eliminates further activation of GCN2 kinase function, and both elF-2a phosphorylation and translation of GCN4 mRNA return to basal levels.

in the absence of amino acid starvation or a tRNA-charging defect (Figure 4) and because gcn gc&-7 double mutants are lethal but can be rescued by the GCNP allele. We propose that the inviability of a gcn4 gcd5-7 double mutant (Niederberger et al., 1988) results directly from the absence of GCN4 protein and the consequent failure to express gcd5-7 at a level sufficient to provide enough LysRS activity for growth. The lethality of gcnl gcd5-7, gcn2gcd57, and gcn3 gcd5-7 mutants would result from the failure to derepress GCN4 protein levels in response to reduced charging of tRNAL*, as GCNl, GCNP, and GCN3 are each required for increased translation of GCN4 in amino acid-starved cells (reviewed in Hinnebusch, 1990). An important corollary of our results is that the GCN2 protein kinase mediates increased translation of GCN4 in response to increased levels of uncharged tRNALfl. It is known that GCNP is required for derepression of GCN4 expression in histidine-starved (Hinnebusch, 1984) and tryptophan-starved cells (Williams et al., 1989). In ad-

Yeaat Stralns and Ganetlc Manlpulatlons Strains used in this study are listed in Table 1. Strain RH776 (Niederberger et al., 1966) the source of the gcd5-7 allele in this study, was crossed with GCDS gcnZ::LEU2 /eu2-3,-772 strain H751, and tetrad analysis of the resulting diploid was conducted, using the Slg- and 5-W phenotypes of gcd5-7, to follow its segregation in the cross. No Leu+ Slg- 5-FT’segregants were obtained, as expected if gcn2::LEU2 gcd5-7 double mutants are inviable (Niederberger et al., 1966). A go&-l segregant of this cross was selected, and repeated backcrosses to H751 were conducted, generatinggcd57 strains JKLlOdA and JKLl958. Strains T66A and T66B were constructed similarly. Strains H750 and H751 were ascospore segregants obtained from a ura%52/ure3-52 /e&3,-7 72//eu2-3,-772 diploid strain in which one copy of GCN2 was replaced with a gcn2::LEU2 deletiondisruption allele, exactly as described in Paddon and Hinnebusch (1969). Strain H397 was constructed by transformation of H15 to Ura’ with plasmid ~139 (Hinnebusch, 1965) a URA3 integrating plasmid containing the GCN4c allele in which all four uORFS have been deleted. p139 DNA was digested with Smal to direct its integration to um3-52. Strains JKL23-1 B and JKL231C were ascospore segregants of a diploid produced by crossing H397 with H750. To test whether inviable gcn god57 double mutants could be rescued by introduction of a GCN4’ allele, we mated the gcd5-7 leu2 mutant JKLl9-5A with strain JKL23-1C containing gcn2::LEUP leu2 and the GCN4C allele integrated at ufa3-52 on a URA3 integrating plasmid. In a control cross, JKLW-5A was mated with JKL23-IB containing gcn2::LEUP leu2 and ura3-52 but lacking the GCNe URA3 plasmid. Both diploid strains were sporulated and subjected to tetrad analysis. In 19 tetrads obtained from the control diploid lacking the GCN4’allele, we obtained no Leu’ Slg- 5-FT’segregants indicative of gcd5-7 gcnP::LEUZ double mutants. In addition, we could deduce that such double mutants accounted for most of the 38% of the ascospores in these tetrads that were inviable. In contrast, the 19 tetrads obtained from the diploid containing a GCN4c allele integrated at ~3.52 contained seven spores that had the Leu’ Slg- 5-FT’phenotypes expected for gcd5-7 gcnP::LEU2 mutants; all seven were Ura’, indicating the presence of the GCN4’allele. In addition, only 16% of the ascospores in these tetrads were inviable, a value close to the 13% inviability expected if the GCN4c allele overcomes the lethal effect of combining gcd57 and gcnP::LEU2. In addition, we tested whether gcn gcd5-7 double mutants could lose a plasmid containing a dominant-constitutive GCN4 allele. To address this, the gcd5-7 leu2 ~3-52 mutant strain T66D, bearing a low copy number URA3 plasmid containing the GCN4A allele, was crossed with strain T61 B containing gcnP::EU2 leu2 ura3-52. GCN4A is missing part of its mRNA leader sequence containing the four uORFs and is equivalent to the GCN4Oallele described above (Hinnebusch, 1965; Mueller and Hinnebusch, 1966). The resulting diploids were sporulated, and 20 tetrads were analyzed, yielding four Slg- 5-FTr Leu’

Table 1. Yeast Strains Used in This Study Strain

Genotype

Source

RH776 JKLl9-5A JKL19-58 JKL23-1 B JKL23-IC

MATa gcd51

P. Niederberger This study This study This study This study

JB32-1 B H397 f-f846 H750 H751 HI5 GRF16 T150A T80A M188D T66A T86B T68D T89B T89A T89C T8lB

met8-1 MATa gcd51 IauZ-3,-112 ura3-52 MATa gcd51 IeuZ-3,-112 ura3-52 MATa gcn2::LEUZ IeuZ-3,-112 ura3-52 MATa gcnP::LElJZ IeuZ-3,.112 ura3-52 GCN4YlRA3 MATa IeuZ-3,-112 ~3-52 GCDC::URAS MATa gcn2-1 ura3-52 IeuZ-3,-112

GCN4aC.URA3 .. MATa gcn3::LElJZ MATa gcn2::LEUZ

This study This study

IeuZ-3,-112 ura3-52 IeuZ-3,112 ura3-52 MATa gcn2::LEM IauZ-3,-112 ura3-52 MATa gcn2-1 ura3-52 IeuZ-3,-112 MATa his3-11,.15 IauZ-3,-112 MATa ura552 his3 MATa GCN4c ade2 trplA1 IeuZ-3,-112 ura552 MATa gcn4 ade2 trplA1 leu2-3,-112 ura3-52 MATa gcd5-1 ura3-52 IeuZ-3,-112 MATa gcd5-1 ura3-52 IeuZ-3,-112 MATa gcd5 1 ura3-52 IeuZ-3,- 112 MATa gcd5- 1 ura3-52 IeuZ-3.-112 MATa gcd5-1 ura3-52 IeuZ-3,-112

MATa gcd5-1 ura3-52 IeuZ-3,-112 MATa gcn2::LEUZ IeuZ-3,-112 ura3-52

Hinnebusch, 1984 This study This study Hinnebusch, 1984 G. Fink This study This study This study This study This study This study This study This study This study This study

trplA1

T61A

MATa gcn3::LEUZ

IeuZ-3,-112

ura3-52

This study

For details of strain constructions, see Experimental Procedures.

segregants presumed to be gcd5-7 gcn2::LEUZ double mutants. All four of these segregants were Ura+, indicating the presence of the GCN4A allele on the URA3 plasmid. None of these spore clones lost the Ura+ phenotype after many generations of nonselective growth on nutrient-rich (YPD) medium, whereas GCD5 gcn2::LEUZ strains bearing the same GCN4c URA3 plasmid readily gave rise to Ura- mitotic segregants under the same conditions. Similar results were obtained when we mated the gcn3::LEUZ leu2 ura3-52 mutant T6lA with strain T68D, which is gcd5-1 leu2 ure3-52 and contains the plasmid bearing GCN4A and URAS. Again, none of three Slg- 5-FT’ Leu’ Ura’ segreg ants obtained in 12 tetrads from this cross lost the Ura+ phenotype when grown for many generations on nonselective medium. These results suggest that the GCN4A allele contained on the autonomously replicating URAJ plasmid is required for the viability of the gcd5-1 gcn2::LEM or the gcd5-7 gcn3::LEUP double mutants. Strain T80A was constructed as described above for JKL23-IC and contains the identical GCN4’allele. Ml 86D, a strain with a mutant gcn4 allele, was obtained by screening for revertants of the Slg- phenotype of the GCN4Oallele in T60A. caused by constitutive high level expression of GCN4. Slg+ revertants were tested for 3-aminotriazole sensitivity (3-AT’), a characteristic of gcn mutants, and for linkage to URA3, since GCN4 is closely linked to URA3. In tetrad analysis of diploids obtained by mating the Slg’ 3AT’ revertant of T60A (M188D) with a URA3 wild-type strain, 3-AT’ was closely linked to Ura+, and none of the ascospores showed a slow.growth phenotype. In addition, when Ml86D was crossed with a gcn4 strain, the 3-AT* phenotype was not complemented. Thus, we concluded that strain Ml86D contains a nonfunctional gcn4 allele. Strains T81 B and T61 A were created by crossing gcn2::LEUZ strain H750 or gcn3::LEUZ strain H646, respectively, with various strains in our collection. T86D and T89B were constructed by transformation of T86A or T868, respectively, with plasmid ~203 (Mueller and Hinnebusch, 1966) a low copy number URA3 plasmid carrying the GCN4Aallele, in which part of the GCN4 leader, including all four uORFs, has been deleted. Strains T89A and T89C were

obtained bytransformingstrainsT86AorT86B, respectively, with plasmid ~232, a low copy number URAI plasmid carrying the GCN4a4 allele, in which upstream AUG codons 3 and 4 have been removed by site-directed mutagenesis(Mueller and Hinnebusch, 1986). This allele, which yields Qfold less GCN4 expression than GCN4A,could also rescue gcd57 gcn double mutants. isolation of the GCDS Gene GCD5 was isolated from a yeast genomic library (Rose et al., 1967) containing 15-20 kb fragments inserted in the low copy number URA3 plasmid YCp50 (Johnston and Davis, 1964) by screening 8000 Ura+ transformants of gcd5-1 strain JKL19-5A for complementation of its slow-growth (Slg-) and 5-FT phenotypes. Plasmids pJBl1 and pJBl2 were recovered from Ura+ Slg+ 5-FT-sensitive yeast transformants by isolating total DNA (Sherman et al., 1968) from the two yeast transformants and using it to transform E. coli to ampicillin resistance. pJBl1 and pJB12 fully complemented the mutant phenotypes of gcd5-1 when reintroduced into strain JKLl9-5A. Restriction site mapping of the two plasmids indicated that the genomic insert in pJBl2 was a subset of the sequences present in pJBl I. Thus, pJBl2 was chosen for further study. To establish that pJBl2 carried the GCD5 gene, we subcloned a portion of the insert from pJBl2 into an integrating URA3 vector and directed integration of the resulting plasmid into the yeast genome at the site homologous to the cloned sequences. Genetic analysis of this transformant revealed that the integrated URA3 marker on the plasmid was tightly linked to the gcd5-7 mutation, thus confirming that pJBl2 contains the GCD5 gene. The integrating URA3 plasmid pJB20 was constructed (see below), containing the 2.4 kb Sal1 fragment from the right end of the genomic insert in pJB12. pJB20 was digested at the unique Bglll site in the genomic sequences and used to transform the GCD5 gcnP:LEUZ ura3-52 strain H750 to Ura+. This Ura+ transformant was crossed with JKLl05A, and the resulting diploid was sporulated and subjected to tetrad analysis, yielding the Ura+ Leu- ascospore segregant JB32-IB containing the inte-

Autoregulation 655

of GCD5 by the General Control System

grated URA3 marker but lacking gcn2AEU2. To test linkage of the URA3 marker with gc&1, JB32-18 was crossed with g&T-7 ~83-52 strain JKLl9-58, and the resulting diploid was subjected to tetrad analysis, scoring gcd57 by its Slg- and 5FT’phenotypes. From 24 tetrads analyzed, we observed 22 parental ditypes, 2 tetratypes, and no nonparental ditypes for the Ura- and Slg--5FT’ phenotypes, suggesting that the URA3 marker had integrated approximately 4 CM from gcd5-7. Based on these results, we concluded that the cloned sequences derive from the GCD5 locus.

Plasmid Constructions GCrV4-/acZ and GCN4”-/acZ fusion constructs on plasmids ~164 and ~227, respectively, have been previously described (Mueller and Hinnebusch, 1986). The integrating plasmid pJB20 was constructed by inserting the 2.3 kb Sal1 fragment from pJB12 into the Sall site of pRS308 (Sikorski and Hieter, 1989). Plasmids containing subclonesof the genomic insert in pJBl2 were generated as follows. pJB48 and pJE49 were constructed by inserting, respectively, the 1.8 kb or 3.7 kb Hindlll fragments from pJB12 into the Hindlll site of the low copy number URA3 plasmid pRS316 (Sikorski and Hieter, 1989). pJB51 and pJB52 were constructed by inserting, respectively, the 3 kb BamHl fragment or the 2.4 kb Sal1 fragment from pJB12 into the BamHl or Sal1 sitesof pRS316. A2.1 kb Bglll fragment from pJB12 was inserted into the BamHl site of YCp50 to create pSL13. Analysis of these subclones for the ability to complement the slow-growth phenotype of gcd5-7 indicated that the sequences required for complementation spanned an internally located -2.5 kb fragment. A GCDS-/acZ fusion was constructed in which IacZ-coding sequences were inserted at codon 69 of the GCD5 ORF, retaining all GCDS sequences both 5’and 3’to the insertion site. This fusion, present on the low copy number URA3 plasmid YCp50, was generated by first deleting the2.4 kb Sal1 fragment from pJB12, resulting in pJB47. A 3 kb /acZ BamHl fragment, obtained from plasmid ~180 (Hinnebusch, 1985), was inserted into the single BamHl site of pJB47, resulting in pSLlPAS-IacZ; the original 3’ terminus of GCDS was restored by inserting the Sal1 fragment from pJBl2 into the unique Sal1 site of pSLlPAS-IacZ, yielding pSL12lacZ.

RNA Analyals DNA restriction fragments for RNA blot hybridization analysis were obtained by digesting pSL1 PlacZ carrying GCDS-/acZwith Clal or plasmid ~227 (Hinnebusch, 1985) containing GCN4-lacZ, with EcoRl and isolating the desired fragments by agarose gel electrophoresis. DNA was isolated from the gel slices using the Prep A Gene purification kit (Bio-Rad). Fifty nanograms of DNAwas labeled with [a-“PJdCTP (6000 Cilmmol, Amersham) using an oligolabeling kit (Pharmacia). The labeled DNA fragments were separated from unincorporated nucleotides by fractionation on a 1 ml Sephadex G25 column. Total yeast RNA was isolated using a heat-freeze method in the presence of phenol and SDS(Schmittetal., 1990); IO-20 pgof RNAwasseparated by agarose-formaldehyde gel electrophoresis and stained with ethidium bromide. Equal amounts of RNA were loaded per lane as monitored by visual inspection of the gel under ultraviolet light. RNA was blotted to Hybond-N membranes, hybridized with radioactively labeled DNA, and washed according to the manufacturer’s recommendation (Amersham). Blots were exposed to X-ray film for 12 to 48 hr at -70°C, using an intensifying screen. The intensity of individual bands was calculated by densitometric analysis (Desago CD60).

In Vitro LysRS Assays Exponentially growing yeast cells were harvested at an OD, of 1 .O1.5 by centrifugation for IO min at 5000 x g, washed two times in buffer E (100 m M Tris-HCI [pH 8.01, 10 m M MgCl>, 1 m M dithiothreitol) and resuspended in buffer E (1 ml per g of wet cells) containing 1 m M phenylmethylsulfonyl fluoride. The suspension was chilled on ice, and cells were broken with glass beads (1 g/ml suspension) by 8 strokes of 20 s each in a Braun MSK Cell Homogenizer. The resulting extract was centrifuged for 15 min at 10,000 x g. The supernatant was analyzed for LysRS activity by aminoacylation of tRNA as previously described (Cirakoglu and Wailer, 1985) with slight modifications: to measure the specific LysRS activity and the total amino acid-charging activity, respectively, the assay contained limiting amounts of extract

(2-10 ug of total protein), 20 m M imidazole-HCI (pH 7.5), 80 m M KCI, 5 m M MgC12, 0.5 m M dithiothreitol, 60 HIM lysine, 3.5 mg of total yeast tRNA (Boehringer), 3 m M ATP, and either 1 t&i of 3H-labeled L-lysine (90 Cilmmol; Amersham) or “C-protein hydrolysate (57 mCi/milligramatom carbon, Amersham), respectively, in a total volume of 50 ~1. To rule out the possibility that dissimilar pool sizes of amino acids from extracts of wild-type and gcd57 mutant cells falsified the results, the extracts were dialyzed and assayed for total tRNA charging as above. The results from dialyzed extracts were identical to those from undialyzed extracts. To determine the saturation curves for ATP, total tRNA, and lysine, assays were conducted as above, except that the corresponding substrate was varied over a range of 0.2-10 x K,.,. Each measurement was done at least in triplicate. Incubation was performed for 10 min (wild type) or 45 min (gc&5-7 mutant strain) at 25OC; charged tRNA was precipitated with cold 5% TCAlO.2% (w/v) lysine, washed three times with 5% TCA/O.2% lysine, dried, and counted in a liquid scintillation counter. To verify that the filter-bound radioactivity originated from lysyl-tRNA, filters were boiled in 5% TCA and examined for retention of the radioactivity. We observed that 95% of the radioactivity was lost, as expected for lysine esterified to tRNA but not for lysine incorporated into protein (data not shown). We also tested the ability of the radioactive material to bind to DEAE-cellulose. Ninety percent of the counts per minute loaded on a DEAE-cellulose column remained bound in 0.3 M KCI (pH 7.5) but eluted from the column in 1 M KCI. This elution behavior is typical for tRNA but not for the vast majority of proteins (data not shown). Taken together, these data indicated that the TCAprecipitable counts per minute generated in our assay stem from lysyl-tRNA.

@Galactosldase Assays Yeast transformations were performed according to the lithium acetate method of Ito et al. (1983). Growth of yeast strains under nonstarvation and starvation conditions and 5-galactosidase assays were performed essentially as described (Mueller and Hinnebusch, 1986). Arginine (2 mglml) and lysine (3 mg/ml) were supplied where indicated. The activities shown are the averages of two independent transformants measured at least twice and have standard deviations of less than

25%. Sequencing of the Mutant gcdS.1 Allele A method employing the PCR (Gyllensten and Erlich, 1988) was chosen to sequence the gcd5-7 allele. Two primers, OPCRl (5’-GCCTCGAGTATTGCTCTGTCCTTCCTCC-3’, corresponding to positions -50 to -31; letters in bold are GC clamp and an additional Xhol site) and OPCRP (5’-GGCTCGAGAAGCTCCTTTAGGGCTACCG-3’; positions 1891-l 872) encompassing the GCD5 ORF and 30 or 92 nt of the 5” and 3’~untranslated region, respectively, were used in equimolaramounts in an initial PCR toamplifythegcd5-7 allele from genomic DNA; theassaycontained, in 100 ul, 50 m M KCI, IOmM Tris-HCI (pH 8.5) 2 m M MgCI,, 50 uM dNTPs, 25 pmol of each primer, 2 U of Taq polymerase (Perkin-Elmer/Cetus), and 7 pg of genomic DNA, which had been denatured for 7 min at 94OC. Cycling parameters were 30 s of denaturation at 94OC, 30 s of annealing at 55OC, 1 min extension at 72OC, and 30 cycles in a Perkin-Elmer DNA thermal cy cler; l/20 of the reaction was analyzed on a 1% agarose gel. One to five microliters (40 ng) of this amplified DNA was used in a second asymmetric PCR, with primer OPCRl present in limiting amounts (0.5 pmol), while primer OPCRP was kept at 50 pmol. Therefore, after IO20 cycles, OPCRl was exhausted so that only minus strand DNA, primed by OPCRP, was newly synthesized. After ethanol precipitation, 20%-50% of this single-stranded enriched DNA was used directly as templateforSangerdideoxysequencing(Sanger et al., 1977) using the Sequenase kit (US Biochemical). The following sequencing primers in addition to OPCRl were used (positions are numbered with respect to the A of the GCDB start codon set to 1): OSLl (5’-CTGGATCCATCGCAAd’, 202-216); OSL2 (5’-GTTCAATTGATGTCCCA-3’, 454-

470); OSL3 (5’~CCAGATACAGAAAGCG-3’ 663-696); , OSL4 (5%GGTGGIlTGGATCGS’, 936-950); OSL5 (5’~CTAGACCATGGAAGAGAA3’, 1175-l 192); OSL6 (5’-GGCCACCCTCAAATG-3’, 1399-1413); OSL7 (5’-CGGTTTACCACCAAC-3’, 1626-l 640). As a control for mutation(s), wild-type DNA was analyzed in the same way.

Cdl 656

Acknowledgments We thank Peter Niederberger and Ralf Huetter for providing us with a gcd5l mutant. This work was supported by grant 31-25565.66 to H. T. and P. P. M. from the Swiss National Foundation. 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 16 USC Section 1734 solely to indicate this fact. Received

Hinnebusch, A. G. (1990). Involvement of an initiation factor and protein phosphorylation in translational control of GCN4 mRNA. Trends Biochem. Sci. 75, 146-152. Hinnebusch. translational H. Trachsel,

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April 10, 1992; revised June 6, 1992.

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Autoregulation 657

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