The Yeast Lysyl-tRNA Synthetase Gene

THE JOURNAL 0 1988 by The American Society for Biochemistry

OF BIOLOGICAL CHEMISTRY and Molecular Biology, Inc

Vol. 263, No. 34, Issue of December 5, pp. 18443-18451,1988 Printed in U.S.A.

The Yeast Lysyl-tRNA Synthetase Gene EVIDENCE FOR GENERAL AMINO ACID CONTROL OF ITS EXPRESSION AND DOMAIN STRUCTURE OF THE ENCODED PROTEIN*

(Received for publication, June 8, 1988)

Marc MirandeS and Jean-Pierre Waller From the Laboratoire d’Enzymologie, Centre National de la Recherche Scientifique, 91190 Gif-sur-Yvette, France

The nucleotide sequence of a 3.6-kilobase pair DNA fragment containing the structural gene for yeast cytoplasmic lysyl-tRNA synthetase (KRSl) and its flanking regions was determined. The encoded protein of 67,881 kDa displays a cluster of 11 lysines within a 29-amino acid residue segment at its amino-terminal extremity. Evidence is presented that this segment is responsible for the affinity displayed by the native enzyme toward polyanionic carriers. The transcription initiation sites of the KRSl gene were determined. Upstream from the TATA box, putative control elements corresponding to the concensus sequences for the RPG box and the general amino acid control system were identified. Evidence for transcriptional induction of the KRSl gene via the general amino acid control system is presented.

We previously reported (Cirakoglu and Waller, 1985a) that yeast aminoacyl-tRNA synthetases, like the corresponding enzymes from higher eukaryotes (Alzhanova et al., 1980, 1982), but unlike those of prokaryotic origin, display the property of binding to polyanionic carriers through ionic interactions. The behavior of purified yeast lysyl-tRNA synthetase was examined in detail (Cirakoglu and Waller, 1985a). It was shown that the native dimeric enzyme (subunit M, 73,000) interacts strongly with immobilized heparin or tRNA as well as with negatively charged liposomes under conditions where the corresponding native enzyme from Escherichia coli ( subunit M, 65,000) does not. Moreover, truncated yet fully active homodimeric forms of the yeast enzyme, with subunit molecular weights of 67,000 and 65,500, respectively, were generated by controlled proteolysis with elastase and papain. The affinity of these modified forms for polyanionic carriers was found to be strongly attenuated in the case of the elastase- modified enzyme and abolished in the case of the papain- modified form. Based on these results, a structural model was proposed according to which each subunit of the native lysyl- RNA synthetase from yeast is composed of a functional domain which is similiar in size to the subunit of the corresponding prokaryotic enzyme (Mr CS.OOO), contiguous to a

*This work was supported in part by Grant 90.1821 from the Centre National de la Recherche Scientifique (Action Thimatique ProgrammBe “Organisation et Expression du GBnome”) and by the Fondation pour la Recherche Medicale. 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.

The nucleotide sequence(s1 reported in thispaper hos been submitted to the GenBankTM/EMBL Data Bank with accession number(s) 504 186.

$ To whom correspondence should be addressed.

structural domain of M, -8,000 carrying a cationic net charge responsible for association with polyanionic carriers.

In order to test this model at the molecular level, the cloning and sequencing of the corresponding gene were undertaken. Cloning of the yeast lysyl-tRNA synthetase gene was recently achieved by probing a Xgtll DNA expression library with antibodies directed against the purified enzyme (Mirande et al., 1986). The cloned gene was positively identified as coding for this enzyme by showing that E. coli lysogens harboring the recombinant DNA phage produced an active yeast lysyl- tRNA synthetase displaying the proper size.

In this paper, we report the nucleotide sequence of the corresponding gene. The interpreted protein sequence reveals the presence of a cluster of cationic amino acids (11 lysines and 2 arginines versus 1 glutamic acid) located between residues 33 and 61 from the amino-terminal extremity. The amino-terminal polypeptide sequence of the elastase-modified enzyme was also determined over a length of 20 amino acid residues. The latter sequence precisely matches the stretch from residues 58 to 77 deduced from the DNA sequence, thereby confirming the identity of the cloned gene and locat- ing the elastase cleavage point between Ala“ and Sers8.

These results are discussed in light of the previously established behavior of the native and proteolytically modified forms of yeast lysyl-tRNA synthetase toward polyanionic carriers (Cirakoglu and Waller, 1985a). They confirm our earlier prediction concerning the existence, in yeast lysyl- tRNA synthetase, of an autonomous structural domain rich in cationic residues responsible for binding to polyanions and unrelated to the expression of catalytic activity. Comparison of the known primary sequences of several yeast aminoacyl- tRNA synthetases suggests that a similar structural organization may be a common feature of most of these lower eukaryotic enzymes.

Concerning the regulation of the expression of yeast lysyl- tRNA synthetase, the nucleotide sequence determined 5’ upstream from the first ATG codon of its structural gene harbors several putative regulatory elements. A RPG box, responsible for coordinate expression of ribosomal protein genes in yeast (Leer et al., 1985), and nucleotide sequences relevant to the general amino acid control system in yeast (Jones and Fink, 1982) were identified. It is shown that transcriptional induction of KRSl mRNA levels via the general amino acid control system is accompanied by translational repression of the expression of the corresponding gene product.

EXPERIMENTAL PROCEDURES

Materials-Restriction endonucleases and DNA-modifying enzymes were purchased from Boehringer Mannheim, Appligene, and New England Biolabs and used as recommended by the suppliers. Radionucleotides and [‘4C]lysine were from Du Pont-New England Nuclear and Commissariat a 1’Energie Atomique (Saclay, France), respectively.

18443

18444 structure of the Yeast Lysyl-tRNA Synthetase Gene

TABLE I Bacterial and yeast strains used in this study Strain Genotype

Bacteria JMlOlTr A(lac,pro), supE, thi, recA56,

srl30O:TnlO (F'traD36, proAB, lacP, hcZAM15)

rsraD139, strA, thi, hfl- A150 [chr::TnlO]

BNN103 AlacU169, proA+, Alon,

Y 1089 BNN103 (pMC9)

Yeast FLlOO C1278b MG409

Wild-type MAT-a Wild-type MAT-a argl1*

Homogeneous lysyl-tRNA synthetase from Saccharomyces cereuisiae and the corresponding antibodies were as previously described (Cirakoglu and Waller, 1985a; Mirande et al., 1986). Wild-type yeast genomic DNA and RNA were gifts from D. Thomas and D. Henry. (Laboratoire d'Enzymologie, Centre National de la Recherche Scien- tifique). Bacterial and yeast strains are listed in Table I. Yeast strains were kindly supplied by Y. Surdin-Kerjan (Laboratoire d'Enzymolo- gie, CNRS), JMlOlTr by Y. Mechulam (Laboratoire de Biochimie, Ecole Polytechnique), and BNNlO3 by B. Guiard (Centre de Gbnb- tique Molkulaire, CNRS).

Analytical Procedures-Sodium dodecyl sulfate (SDS)'-polyacrylamide gel electrophoresis was performed according to Laemmli (1970) on 10% acrylamide gels. The protein blotting procedure was conducted as previously described (Mirande et al., 1982) using affinity- purified goat anti-rabbit IgG conjugated to peroxidase (Biosys, Com- piigne, France).

For Northern blot analysis, RNA was subjected to electrophoresis in formaldehyde-agarose gels as described (Maniatis et al., 1982) and blotted onto Genescreen Plus filters (Du Pont-New England Nu- clear), followed by backing for 2 h a t 80 "C. Prehybridization was conducted at 65 "C for 1 h in 50% formamide, 5 X SSPE (900 mM NaCI, 50 mM NaH2P04, 5 mM EDTA, pH 7.4), 5% SDS, 2.5 X Denhardt's solution, 5% dextran sulfate, 150 pg/ml yeast tRNA, and 5 pg/ml poly(A)+, followed by hybridization at 65 "C for 16 h in the same buffer containing lo6 cpm/ml ["PIRNA probes. Filters were washed with 0.1 X SSPE and 0.5% SDS at 65 "C.

Southern blot analysis was performed according to Maniatis et al. (1982) using the same hybridization procedure.

DNA fragments were isolated by high performance size exclusion chromatography (Schmitter et al., 1986).

Protein Sequencing-Homogeneous yeast lysyl-tRNA synthetase (subunit M, 73,000) and the elastase-modified, fully active form (subunit M, 67,000) (Cirakoglu and Waller, 1985a) were subjected to N-terminal amino acid sequence analysis.

Automated Edman degradations were performed by J.-P. Le Caer (Service Commun de Microsbquencage, Laboratoire de Physiologie Nerveuse, Centre National de la Recherche Scientifique) using an Applied Biosystems 470A Sequencer. Phenylthiohydantoin deriva- tives were identified by reverse-phase high performance liquid chromatography.

DNA Sequencing-DNA from XLys8, a derivative of Xgtll carrying a 3.6-kilobase pair yeast genomic insert encoding lysyl-tRNA synthetase in the EcoRI site (Mirande et al., 1986), was purified from induced lysogen Y1089/XLys8 by the procedure of Maniatis et al. (1982). Large fragments of the inserted DNA were subcloned in M13mp18 or M13mp19 DNA (Yanisch-Perron et al., 1985). The complete sequence was determined by the dideoxynucleotide chain termination method (Sanger et al., 1977). Sets of overlapping fragments were obtained by limited Bal31 digestion, followed by end-filling with DNA polymerase I (Klenow fragment). The truncated inserts were excised, treated with calf alkaline phosphatase, separated from the M13 DNA by size exclusion chromatography, and subcloned in suitable non- dephosphorylated M13mp18 or M13mp19 DNA which was made free of its excised polylinker fragment. The ligation mixture was used to transform JMlOlTr.

The abbreviations used are: SDS, sodium dodecyl sulfate; bp, base pair(s); PIPES, 1,4-piperazinediethanesulfonic acid; Ap4A, 5',5'-dia- denosine tetraphosphate; ORF, open reading frame.

DNA and protein sequences were analyzed by using the BISANCE programs from the Centre Interuniversitaire de Traitement de 1%- formation facilities (Paris).

Probe Synthe~is-[a-~~P]UTP-labeled RNAs, obtained by in uitro transcription with bacteriophage SP6 polymerase, were used as probes for Northern and Southern experiments.

The -600-bp EcoRI-EcoRI fragment (positions +387 to +lo13 in Fig. 2) was ligated into the EcoRI site of pSP65 plasmid DNA. The ligation mixture was used to transform BNNlO3. The recombinant plasmid pSP65-YKRS&, corresponding to antisense RNA transcripts from the SP6 promoter, was selected. In uitro transcription was performed as described by Melton et al. (1984).

A -1100-bp HindIII-XhoI fragment of the actin gene of S. cereuis- ine (Gallwitz and Sures, 1980) in plasmid pYA301 (kindly supplied by D. Thomas) was subcloned in HindIII-SalI-digested pSP64 DNA. In uitro transcripts from pSP64-YA- plasmid correspond to antisense RNA probes.

RNA Preparation-Total RNA from yeast was isolated from strains FLlOO and 21278b grown in YNB medium (0.7% Difco yeast nitrogen base supplemented with 2% glucose) and from strain MG409 grown in YNB medium supplemented with arginine at 1 mg/ml for repression or 10 pg/ml for derepression of amino acid biosynthetic enzymes following the procedure described by Cherest et al. (1985). Poly(A)+ RNA was isolated by chromatography on oligo(dT)-cellulose as described by Maniatis et al. (1982).

SI Nuclease Mapping-Twenty-five micrograms of poly(A)+ RNA from yeast strain FLlOO grown in YNB medium was mixed with 5 X 10' cpm of a 5'-end-labeled probe corresponding to a BsmI-HpaII fragment (positions -272 to +68 in Fig. 2). The mixture was ethanol- precipitated, resuspended in 30 p1 of hybridization buffer (40 mM PIPES (pH 6.4), 400 mM NaCl, 1 mM EDTA, 8 0 % formamide), heated at 90 "C for 5 min, and incubated at 42 'C for 4 h. The hybridization mixture was quickly transferred into 300 pl of ice-cold S1 buffer (50 mM sodium acetate (pH 4.6), 280 mM NaCl, 4.5 mM ZnS04, and 20 pg/ml sonicated salmon sperm DNA). Digestion was conducted at 30 'C for 60 min with 150 units of S1 nuclease (Boeh- ringer Mannheim) and stopped by the addition of 10 pl of 0.5 M EDTA (pH 8.0). The mixture was ethanol-precipitated, washed with ethanol, and resuspended in 10 pl of standard formamide loading buffer. The DNA probe used for S1 mapping was also subjected to G + A and T + C sequencing reactions according to Maxam and Gilbert (1980).

Assay of Lysyl-tRNA Synthetase-Exponentially growing yeast cells (in 500 ml of liquid medium; A m - 1-1.5) were harvested by centrifugation (5,000 X g, 10 min), washed twice in extraction buffer (100 mM Tris-HC1 (pH S.O), 10 mM MgC12, 1 mM dithioerythritol), and resuspended in extraction buffer (1 ml/g of cells) containing 1 mM diisopropyl fluorophosphate and 1.2 mM phenylmethylsulfonyl fluoride. The cell suspension was quickly frozen in Eaton press blocks kept in dry ice, and cells were broken at a pressure of 5,000 p.s.i. The resulting extract was centrifuged for 15 min at 10,000 X g. The supernatant was assayed for lysyl-tRNA synthetase activity by the aminoacylation of tRNA as previously described (Cirakoglu and Waller, 1985a). One unit of activity corresponds to 1 nmol of aminoacyl-tRNA formed per min at 25 "C. Protein concentration was determined according to Gornall et al. (1949). L-Ornithine carbamoyltransferase was assayed according to Ramos et al. (1970).

RESULTS

Nucleotide Sequence of Yeast Genomic DNA Insert from Uys8 Encoding Lysyl-tRNA Synthetase-A X g t l l recombinant DNA clone, XLys8, allowing the expression of a native lysyl-tRNA synthetase from yeast origin was previously isolated by probing a DNA expression library with antibodies directed against the purified enzyme (Mirande et al., 1986). The location of the KRSl gene, corresponding to yeast lysyl- tRNA synthetase, was determined by the characterization of two other recombinant clones, one of them allowing the expression of a P-galactosidasellysyl-tRNA synthetase fusion protein (Mirande et ai., 1986). In this work, the nucleotide sequence of the 3.6-kilobase pair DNA insert from XLys8 is established. The complete DNA insert was sequenced on both strands by the strategy shown in Fig. 1. Generation of overlapping fragments by Ba131 deletions represents a very con- venient and rapid procedure for sequencing large fragments.

Structure of the Yeast Lysyl-tRNA Synthetase Gene 18445

FIG. 1. Restriction map and sequencing strategy of KRSl gene and its flanking regions. The restriction map shows only relevant sites for EcoRI ( E ) , BsmI (Bs), BanHI (Ba), XbaI ( X ) , HindIII ( H ) , and Sac1 ( S ) . The EcoRI sites at the 5’- and 3’-ends of the yeast insert are contributed by EcoRI linkers. The protein coding regions are indicated by solid bars, and the arrows indicate the sense of transcription (from left to right for the KRSl gene and from right to left for the unidentified open reading frame ORF2). The complete overlapping sequence was determined by the dideoxynucleotide chain termination method (Sanger et al., 1977). The arrows indicate the direction and extent of sequence determination. kb, kilobase.

A long open reading frame of 1773 bp, corresponding to the KRSl gene, was found (positions +1 to +1773 in Fig. 2). In addition, a second open reading frame was observed 726 bp upstream from the initiation codon of the KRSl gene. These two transcription units present opposite polarities. By using a 600-nucleotide-long anti-RNA probe corresponding to the internal EcoRI-EcoRI fragment of the KRSl gene, a unique polyadenylated mRNA of 1900-2000 nucleotides was detected by Northern blot analysis (Fig. 3). That the putative ORF2 gene actually encodes a protein is supported by the observation that a polyadenylated mRNA of 1700-1800 nucleotides can be detected by using a 1150-nucleotide-long anti-RNA probe corresponding to the EcoRI-EcoRI fragment encompassing the 3‘-end of the DNA insert shown in Fig. 1.

The KRSl translation unit has a codon bias index of 0.54, characteristic of moderately expressed proteins (Bennetzen and Hall, 1982). The corresponding value for the fragment of the unidentified ORF2 gene is 0.17, suggestive of a regulatory, low abundance protein.

Characteristic Features of KRSl Gene Product-The predicted protein encoded by the KRSl gene is 591 amino acid residues in length, with a calculated molecular weight of 67,881, in reasonable agreement with that estimated by SDS- polyacrylamide gel analysis of the purified yeast cytoplasmic lysyl-tRNA synthetase (subunit M , 73,000).

It was previously shown that the native lysyl-tRNA synthetase can be converted to a fully active modified dimer of subunit M, 67,000 upon elastase treatment (Cirakoglu and Waller, 1985a). These two homogeneous forms were subjected to automated Edman degradations. Analysis of the native enzyme reveals the presence of a blocked N-terminal residue. Taking in account the observed patterns of the effect of the penultimate amino acid on post-translational modifications in yeast (Huang et al., 1987) showing that methionine is removed and that serine is blocked when the penultimate residue is a serine, this result is in accordance with the N- terminal amino acid sequence determined for the KRSl gene product: Met-Ser.. . Treatment of the native enzyme with elastase leads to exposure of a single free N-terminal residue. The following amino acid sequence was obtained N-Ser-Lys- Lys-Lys-Thr-Asp-Leu-Phe-Ala-Asp-Leu-Asp-Pro-Ser-Gln- Tyr-Phe-Glu-Thr-Arg. . . , corresponding to residues 58-77 of the predicted protein encoded by the KRSl gene. This result confirms the unambiguous assignment of the DNA insert to

the KRSl gene, previously based on the observation that the XLys8 DNA insert allows the expression of an active yeast lysyl-tRNA synthetase in E. coli lysogens (Mirande et al., 1986).

It was previously shown that the aptitude of the native lysyl-tRNA synthetase to interact with polyanionic carriers in uitro was strongly decreased on elastase conversion to a fully active modified dimer of subunit M, 67,000 and was completely abolished on papain conversion to a fully active enzyme of subunit M, 65,500 (Cirakoglu and Waller, 1985a). Knowledge of the protein sequence of lysyl-tRNA synthetase provides a rational explanation for this behavior. Cleavage by elastase leads to deletion of an N-terminal fragment of 57 amino acid residues comprising a cluster of 8 lysines located between residues 33 and 55, with consequent attenuation of the affinity of the truncated enzyme for polyanionic carriers. Upon further elimination of lysine residues 59-61 by papain treatment, which is expected to remove approximately 10 additional residues (MI 67,000 + 65,500), this residual affinity is abolished. From the amino acid sequence of the KRSl gene shown in Fig. 2, an isoelectric point of 6.36 can be calculated for the native full-length monomer of M , 67,881. Similarly, a PI of 5.79 for the elastase-modified enzyme (residues 58-591) and of 5.64 for the papain-modified enzyme (taking into account residues 67-591) can be determined. Correspondingly, the polypeptides from residues 1 to 57 and 1 to 66 display PI values of 10.09 and 10.16, respectively.

Taken together, these results strongly support the earlier proposal according to which each subunit of yeast lysyl-tRNA synthetase is composed of a functional domain which is similar in size to the subunit of the prokaryotic enzyme, contiguous to a binding domain responsible for association to negatively charged carriers (Cirakoglu and Waller, 1985a), the latter being localized in the N-terminal part of the molecule. The significance of this finding will be further addressed under “Discussion.”

Characteristic Features of KRSl Transcription Unit-In order to determine whether or not lysyl-tRNA synthetase from yeast is encoded by a single nuclear gene, Southern analysis of total genomic yeast DNA was performed. As shown in Fig. 4, by probing with the RNA transcript from pSP65-YKRS&, unique DNA fragments of 600, 2100, 3700, and 5000 bp were detected following digestion with EcoRI, BglII, BamHI, or HindIII, respectively. Whereas the EcoRI

18446 Structure of the Yeast Lysyl-tRNA Synthetase Gene

FIG. 2. Nucleotide sequence of 3.6-kilobase pair yeast DNA insert containing KRSl gene and deduced amino acid sequences. The nucleotide sequence of the sense strand corresponding to the KRSl gene open reading frame is indicated by upper-case letters. Its deduced amino acid sequence from nucleotides +1 to +1773 is indicated under the DNA sequence. Amino acid residues 1-57, removed by elastase treatment of the purified enzyme, are underlined; and lysine residues are indicated by asterisks. The putative TATAA transcriptional initiation signal (position -75) is underlined, and the RNA initiation sites (positions -23, -32, -43, -49, and -69) are ouerlined. The sequence 5"TCCTTCC- 3' (positions -40 to -34), complementary to the 3'-end of 18 S ribosomal RNA (3'-AGGAAGG-5'), is underlined. The RPG box (positions -227 to -238) is indicated by an arrow, and the consensus sequences for general amino acid control (located between positions -188 and -129) are underlined. The tripartite consensus sequence for transcription termination (TAG.. .TATGA.. .TTT) and two putative AATAAA polyadenylation sites located in the 3'-flanking region are underlined. The deduced amino acid sequence of the nonsense strand corresponding to the unidentified open reading frame (ORF2) from positions -727 to -1522 is shown above the DNA sequence. Its putative TATAA box at position -693 is underlined.

fragment is internal to the K R S l gene, digestion with BglII or BamHI gives rise to large fragments encompassing the 3'- flanking regions of the K R S l gene, and digestion with HindIII corresponds to its 5"upstream sequence. These results indicate that yeast cytoplasmic lysyl-tRNA synthetase is encoded by a unique nuclear gene.

The mRNA initiation site of the KRSl gene was determined by S1 nuclease protection experiments. As shown in Fig. 5, five major initiation sites were found at positions -69, -49, -43, -32, and -23 upstream from the translation initiation

codon. A Goldberg-Hogness TATAA box, corresponding to the consensus sequence for the transcription initiation signal (Chen and Struhl, 1985) is located at positions -75 to -71 (Fig. 2). At positions -40 to -34, a 7-nucleotide-long sequence (5'-TCCTTCC-3') is exactly complementary to the 3'-end of the 18 S ribosomal RNA from yeast (3'AGGAAGG-5'; Rubt- sov et al., 1980). The primary structure of the 3'-end of 18 S ribosomal RNA from eukaryotic cells is highly conserved, suggesting that an mRNA-rRNA interaction may contribute to mRNA recognition by eukaryotic ribosomes (Hagenbiichle

Structure of the

1 2 3 1 2 3

- 4713 - 3392

Yeast Lysyl-tRNA Synthetase Gene 18447

G T + + 1 2 A C

A B

FIG. 3. Identification of KRSI and ORF2 transcripts. Poly(A)+ mRNA (lane I, 2 pg; lane 2, 1 pg) or total cellular RNA (lane 3, 10 pg) was run in a 1% agarose gel containing formaldehyde. Northern blot analysis was conducted using the labeled SP6 transcripts corresponding to anti-KRSI mRNA (A ) or anti-ORFZ mRNA (R). Ribosomal RNAs from Chinese hamster ovary cells (4713 and 1879 nucleotides) and yeast (3392 and 1789 nucleotides) were used as internal markers.

A B C D

sooo- 3700 - 2100 -

800-

3 * V

c)

FIG. 4. Southern hybridization analysis of yeast genomic DNA. Total genomic yeast DNA was digested with EcoRI ( l a n e A) , RamHI ( l a n e R) , BglII ( l a n e C), or HindIII (lane D) and subjected to fractionation in a 1% agarose gel. Southern blot analysis was carried out using an RNA probe corresponding to the 600-bp-long internal EcoRI-EcoRI fragment (positions +386 to +lo09 in Fig. 2) of the KRSl gene. The DNA fragments of 3700 and 2100 bp visualized following digestion with BamHI (position +204) or BglII (position +229) encompass the 3”flanking region of the KRSI gene, whereas the 5000-bp fragment maps the 5”flanking region (HindIII, position +1348).

et al., 1978). In the 3”flanking region of the KRSI gene, a sequence (TAG.. .TATGA.. .TTT) homologous to the tripartite consensus sequence required for transcription termination in yeast (Zaret and Sherman, 1982) as well as twice repeated potential eukaryotic polyadenylation signal (AA- TAAA, Proudfoot and Brownlee, 1976) are found downstream from the TAA stop codon. Taking into account the initiation site a t position -49, the polyadenylation signal a t position +1899, and a poly(A) tract of about 50 adenine residues, an mRNA species of -2000 nucleotides is expected, in accordance with the size of the transcript detected in Fig. 3.

-70 - -60 - -50 -

-40 - -30 -

-20 -

-10 -

+t -

+10 -

FIG. 5. Determination of 5’-ends of KRSI transcript. Analy- sis of the 5’-ends of the KRSI transcript by S1 mapping was carried out as described under “Experimental Procedures.” The 5”end-labeled DNA fragment (positions -272 to +68) was subjected to base- specific chemical cleavages (G + A, T + C) or hybridized to poly(A)+ mRNA. Lane 1 , S1 nuclease-resistant fragments; lane 2, BsmI-HpaII fragment used as DNA probe. At left, the numbers refer to the positions in the DNA sequence shown in Fig. 2. At right, the arrows indicate the major S1 nuclease-protected ends. The S1 nuclease- resistant faint band in lane 1 migrating as a 220-bp fragment is a contamination of the probe with the downstream HpaII-EcoRI fragment of KRSZ (positions +167 to +386).

Concerning the nucleotide sequence determined 5’ upstream from the TATA box, several stretches of putative regulatory elements could be observed. Located between positions -238 and -227, a 12-nucleotide sequence (5’- TAGTGTATGGGT-3’), complementary to 5”ACCCATA- CACTA-3’, is closely homologous to the sequence 5‘-

ACCCATACATTd-3’, corresponding to the RPG box which

is supposed to be involved in the coordinate control of ribosomal protein gene expression in yeast (Leer et al., 1985). Such conserved sequences are generally found 200-450 bp upstream from the ATG codon, either on the coding or the noncoding strand of the gene.

In addition, nucleotide sequences which were found to be relevant to the general amino acid control system in yeast (Jones and Fink, 1982) are found in the 5”upstream region c; ;he KRSl gene, encompassing nucleotides -188 to -129. Indeed, it was shown that the short nucleotide sequence 5’- TGACTC-3’ (located between positions -129 and -134 in the KRSI gene; Fig. 2) is required for regulation of several

T


genes involved in the biosynthetic pathways of several amino acids in yeast (Hinnebusch and Fink, 1983; Donahue et al., 1983; Hill et al., 1986; Crabeel et al., 1985). As in the case of the HIS3 gene (Hill et al., 1986), a T-rich sequence precedes the TGACTC box.

General Amino Acid Regulation of KRSl mRNA Levels- Taking into account the putative regulatory element TGACTC, we have investigated the possibility that KRSl gene expression may be under control of the general amino acid system in yeast. A wild-type strain (21278b) and an arginine leaky auxotrophic strain (MG409) were grown in minimal medium supplemented with arginine for repression or starved for arginine for derepression of amino acid biosynthetic enzymes. Northern blot analysis of total RNA was performed to determine the steady-state level of KRSl mRNA. An actin anti-mRNA probe was used as an internal marker. As can be seen in Fig. 6, whereas similar amounts of KRSl mRNA were detected from 2127813 grown in minimal medium and MG409 grown in the same medium supplemented with arginine, an approximately s-fold increase in the KRSl mRNA level was observed from MG409 grown under arginine restriction, with the amount of actin mRNA remaining un- changed. This result strongly argues in favor of general amino acid regulation of KRSl mRNA expression. Lysyl-tRNA synthetase activity was determined in the corresponding crude extracts. As shown in Table 11, no elevation of lysyl-tRNA synthetase activity was observed following induction of its corresponding mRNA in the presence of limiting amounts of arginine in the culture medium. As a control, L-ornithine carbamoyltransferase, an enzyme that is subject to general amino acid control in yeast (Crabeel et al., 1985), was assayed in the same extracts. A %fold increase of L-ornithine carbamoyltransferase activity was observed following induction by

1 2 3

FIG. 6. General amino acid regulation of KRSl mRNA levels. Total RNA (10 pg) from S1278b (lane I ) grown in YNB medium or from MG409 grown in YNB medium containing arginine at 1 mg/ ml (lane 2 ) or 10 pg/ml (lane 3 ) was subjected to Northern blot analysis using 32P-labeled anti-RNA probes corresponding to lysyl- tRNA synthetase ( K R S I ) or actin. By probing with the KRSI probe or the actin probe alone, only the corresponding transcripts were visualized.

TABLE I1 Assay for lysyl-tRNA synthetase and L-ornithine

carbamoyltransferase activities Strain Growth medium (28 'C) KRS" OTCase*

C1278b YNB 1.31 1.00

MG409 YNB + L-arginine (1 mg/ml) 1.30 0.39 YNB + L-arginine (10 pg/ml) 1.48 2.79

"KRS, lysyl-tRNA synthetase. Values are specific activity expressed in nmol of lysyl-tRNA formed per min/mg of protein at 25 "C.

OTCase, L-ornithine carbamoyltransferase. Values are expressed in arbitrary units relative to the value determined in extracts from C1278b.

1 2 3 4

KRS 4 --

FIG. 7. Western blot analysis of lysyl-tRNA synthetase levels in crude extracts from yeast. Crude extracts (5 pg of protein) from 31278b (lane 2 ) grown in YNB medium or from MG409 grown in the same medium supplemented with arginine at 10 pg/ml (lane 3 ) or 1 mg/ml (lane 4 ) were analyzed by the protein blottingprocedure using affinity-purified anti-lysyl-tRNA synthetase antibodies. Lune

73,000). I, 0.025 pg of purified lysyl-tRNA synthetase (KRS; subunit M,

arginine starvation, as compared to the activity determined in the wild-type strain. We have verified by the protein blotting procedure that the polypeptide of M , 73,000, corresponding to lysyl-tRNA synthetase, was not overexpressed in MG409 cells grown in the presence of limiting amounts of arginine (Fig. 7). Thus, the transcriptional induction via the general amino acid control system is counteracted by a post- transcriptional mechanism, leading to translational repression of the expression of lysyl-tRNA synthetase.

DISCUSSION

Yeast Aminoacyl-tRNA Synthetases Are Composed of Two Independent Domains-Lysyl-tRNA synthetases from E. coli and yeast display the same oligomeric structure of the a 2 -

type, yet differ significantly in their apparent subunit molecular weights: 65,000 for the E. coli enzyme (Hirshfield et al., 1976) and 73,000 for the yeast enzyme (Cirakoglu and Waller, 1985a). The chain extension that characterizes yeast lysyl- tRNA synthetase was shown to be responsible for the polyanionic binding property displayed by this enzyme. Whereas native lysyl-tRNA synthetase from yeast (subunit M, 73,000) binds strongly to polyanionic carriers, a papain-modified fully active form (subunit M, 65,500), like the corresponding native enzyme from E. coli (MI 65,000), does not (Cirakoglu and Waller, 1985a). In light of the results reported in this study, the binding property displayed by yeast lysyl-tRNA synthetase can be assigned to the N-terminal moiety of its polypeptide chain. Indeed, among the 66 amino-terminal residues corresponding to the portion of the molecule removed follow- ingpapain treatment, the interpreted protein sequence reveals the presence of 13 lysines and 2 arginines versus 3 aspartic acids and 5 glutamic acids, accounting for a calculated PI of 10.16 versus 6.36 for the native enzyme. Moreover, a cluster of basic residues (11 lysines + 2 arginines) is located between residues 33 and 61. By using the algorithm of Garnier et al. (1978), an a-helix secondary structure can be predicted for the N-terminal part of yeast lysyl-tRNA synthetase. Assum- ing the validity of this prediction, a helical wheel representation for residues 33-60 leads to the striking observation that the lysine residues are distributed on one-half of the helix section (Fig. 8).

The difference in behavior toward polyanionic carriers which characterizes lysyl-tRNA synthetases from E. coli and


yeast appears to be a general property of the aminoacyl-tRNA synthetases from these sources. Whereas prokaryotic aminoacyl-tRNA synthetases do not interact with immobilized polyanionic carriers (Alzhanova et al., 1980), those from lower (Cirakoglu and Waller, 1985a) or higher (Alzhanova et al., 1980; Cirakoglu and Waller, 1985b) eukaryotes do. Compari- son of the primary structures of those seven aminoacyl-tRNA synthetases, the sequences of which are presently known from E. coli as well as from S. cereuisiae (Fig. 9), leads to the following observations. (i) The subunit sizes of the yeast enzymes are invariably larger than those of the corresponding enzymes from E. coli. (ii) Following alignment of their primary structures, the polypeptide chain extensions that characterize the yeast enzymes are found to be located at one extremity of

v 4 1

Kss

K 4 9 7

E 42

FIG. 8. Helical wheel representation of lysine-rich N-terminal domain of yeast lysyl-tRNA synthetase. Amino acid residues 31-60 from yeast lysyl-tRNA synthetase, for which an whelix secondary structure can be predicted, are arranged in a helical wheel representation as described for aspartyl, threonyl-, and valyl-tRNA synthetases from yeast (Lorber et al., 1988).

t HRSEC

1 so HRSSCC

121

52li

the molecules, rather than as insertions within the conserved regions. (iii) These chain extensions display remarkably basic isoelectric points and are generally located in the N-terminal moiety of the molecules, accounting for at least 10% of the primary structures (28% in the case of yeast glutaminyl-tRNA synthetase). One exception is yeast seryl-tRNA synthetase, which displays a C-terminal extension of 13 residues comprising a cluster of 6 lysines. It is noteworthy that a similar feature prevails in the case of all eukaryotic elongation factors la, which harbor C-terminal extensions of 18-20 amino acid residues comprising 7 lysines relative to their prokaryotic or mitochondrial elongation factor Tu counterparts (Brands et al., 1986). It was previously shown that elongation factor la binds to polyanionic carriers under conditions where the corresponding elongation factor Tu from E. coli does not (Domogatsky et al., 1978). However, the relevance of this C- terminal cationic extension to polyanion binding was not tested experimentally. Isoleucyl-tRNA synthetase from yeast also carries a C-terminal extension relative to its prokaryotic counterpart. This stretch is composed of primarily acidic residues (PI 5.00 for residues 930-1073). However, a cluster of 7 lysines, absent from the E. coli enzyme, is present between residues 906 and 916 in the region just preceding the chain extension that characterizes the yeast enzyme. In the case of methionyl-tRNA synthetase, in addition to the N-terminal cationic chain extension found in the yeast monomeric enzyme, a C-terminal extension is found for the E. coli enzyme corresponding to a C-terminal domain implicated in the di- merization of the E. coli enzyme (Cassio and Waller, 1971).

In addition to the seven enzyme couples listed in Fig. 9, the primary sequence of aspartyl-tRNA synthetase from yeast is also available (Sellami et al., 1986). The N-terminal moiety of this molecule likewise displays markedly cationic proper- ties: PI 8.87 for residues 1-100, compared to 6.84 for the native enzyme. Furthermore, helical wheel representations of the lysine-rich N-terminal domains of aspartyl-, threonyl-, and valyl-tRNA synthetases from yeast reveal an anisotropic dis- tribution of the lysine residues (Lorber et al., 1988), as shown here for yeast lysyl-tRNA synthetase (Fig. 8).

Thus, comparison of the known primary structures of E. coli and yeast aminoacyl-tRNA synthetases suggests that a

HRSEC + 424 1.10

HRSSCC 1 - u 6 ?.s 1 ~ 9 0 en 00-u6 8 1 5

? SRSEC

SRSSCC

430

UDm

t VRSEC 050

140 VRSSCC VRSEC 1-95? 5 . U

VRSSCC 1-1101 711 1-140 1 4 0 1 1 0 1 6.06 1 ? 0 1

FIG. 9. Comparison of homologous aminoacyl-tRNA synthetases from E. coli and yeast. Alignment of homologous aminoacyl-tRNA synthetases is based on the Kanehisa alignment program (Kanehisa et al., 1984) by weighting with the mutation data matrix (Dayhoff et al., 1983). Aminoacyl-tRNA synthetases are designated as follows: XRSEC and XRSSCC, aminoacyl-tRNA synthetase specific for the amino acid X (one-letter symbol) from E. coli ( E C ) or S. cereuisiae cytoplasm (SCC). The region of homology is indicated by solid bars, and N- or C- terminal extensions are shown by discontinuous bars. Number I corresponds to the N-terminal amino acid residue. At right, calculated isoelectric points (in bouface) are shown for amino acid residues x to y . Protein sequences are deduced from the nucleotide sequences of the corresponding cloned genes: HRSEC (Freedman et al., 1985), HRSSCC (Natsoulis et al., 1986), IRSEC (Webster et al., 1984), ZRSSCC (Englisch et al., 1987), MRSEC (Barker et al., 1982; Dardel et d., 1984) MRSSCC (Walter et al., 1983), QRSEC (Hoben et al., 1982), QRSSCC (Ludmerer and Schimmel, 1987a), SRSEC (Hartlein et al., 1987b), SRSSCC (Weygand-Durasevic et al., 1987); TRSEC (Mayaux et al., 1983), TRSSCC (Pape and Tzagoloff, 1985), VRSEC (Hartlein et al., 1987a), and VRSSCC (Jordana et al., 1987).


similar structural organization may be a common feature of most, if not all, of the lower eukaryotic enzymes. These observations support the view that lower eukaryotic aminoacyl-tRNA synthetases have evolved from an ancestral enzyme similar in size to that from prokaryotes by acquiring a structural domain conferring to them the ability to bind to polyanionic carriers. As regards the functional significance of this evolutionary acquisition, it was proposed earlier that this binding domain may promote the compartmentalization of these enzymes within the cytoplasm, at or near the site of protein synthesis, through electrostatic interactions with as yet unidentified cellular components carrying negative charges (Cirakoglu and Waller, 1985a). The anisotropic dis- tribution of the cationic charges within the binding domain may be optimally suited to ensure oriented anchorage of these enzymes to polyanionic surfaces.

Tentative experiments were recently carried out to test the significance of the large amino-terminal extension borne by glutaminyl-tRNA synthetase from S. cereukiae (Ludmerer and Schimmel, 198713). No evidence for a specific role was found. However, the internal deletions examined did not concern the entire N-terminal extension of 230 residues. In particular, the 9 lysines located between positions 188 and 204 were present in all of the constructions analyzed.

Cytoplasmic Yeast Lysyl-tRNA Synthetase Is Encoded by Unique Nuclear Gene-Lysyl-tRNA synthetase from E. coli is encoded by two genes: a constitutive gene (lysS) and a heat- inducible gene (lysU) which were mapped at 62.1 and 92 min on the E. coli chromosome, respectively (Emmerich and Hirshfield, 1987; Van Bogelen et al., 1983). By contrast, Southern analysis of total genomic yeast DNA reveals the presence of a single nuclear gene encoding lysyl-tRNA synthetase.

It was shown that lysyl-tRNA synthetase from E. coli, yeast, or sheep liver catalyzes the in vitro synthesis of 5’,5’-diaden- osine tetraphosphate (Blanquet et al., 1983). Several lines of evidence have suggested that this unusual dinucleotide may act as a pleiotropic alarmone (Varshavsky, 1983). Assuming that in uiuo the lysU gene product is implicated in ApA synthesis following the heat-shock response, we have to con- sider that in yeast its synthesis may be ensured in another way. However, the possibility that KRSl gene expression may be heat-inducible or that another form of yeast lysyl-tRNA synthetase may be encoded by a gene harboring too low homology with KRSl to be detected by hybridization cannot be completely dismissed.

Regulation of Expression of Yeast Lysyl-tRNA Synthetase- Whereas structural elements involved in the regulation of the expression of the three bacterial aminoacyl-tRNA synthetases specific for the amino acids alanine (Putney and Schimmel, 1981), threonine (Springer et al., 1985)) and phenylalanine (Fayat et al., 1983) have been identified and characterized, examination of the 5’-upstream regions of the yeast aminoacyl-tRNA synthetase genes sequenced to date has provided little insight on the regulatory mechanisms implicated in their expression. Concerning the expression of yeast lysyl-tRNA synthetase, several putative regulatory elements were identified 5’ upstream from the ATG codon of the KRSl gene.

The genes encoding the majority of yeast ribosomal proteins and elongation factor la where shown to harbor conserved sequences (HOMOL1 and RPG box) in their 5’-flanking regions (Leer et al., 1985). It was proposed that a DNA- binding protein component may be a general factor for transcriptional activation of these genes (Huet et al., 1985). By extension, it may be inferred that the RPG box found in the 5”noncoding region of the KRSl gene allows coordinated regulation of its expression, in relation to a large family of

genes coding for the translational machinery in yeast. Located downstream from the RPG box, sequence elements

relevant to the general amino acid control system were found. It was shown that these regulatory elements alone are able to confer general amino acid control on the E. coli P-galactosid- ase gene (Silverman et al., 1982) or on the yeast cytochrome c gene (Hinnebusch et al., 1985). Moreover, a single mutation in the TGACTC sequence affects the binding of the GCN4 activator protein (Hill et al., 1986). The results presented in this report unambiguously demonstrate that transcription of the KRSl gene is controlled by the general amino acid system. A similar observation was previously reported for the ILSl gene encoding yeast isoleucyl-tRNA synthetase (Meussdoerf- fer and Fink, 1983). The nucleotide sequence of that gene was recently determined (Englisch et al., 1987). In the 5”upstream region of the ILSl gene, a TGACTC sequence can be observed (positions -102 to -97) preceded by a T-rich sequence (positions -151 to -134). However, no data concerning the expression of the ILSl gene product are available. In the case of yeast lysyl-tRNA synthetase, we have observed a post- transcriptional repression of its expression following arginine starvation of a leaky auxotrophic strain. The molecular features responsible for this secondary effect remain to be determined.

Concerning the physiological significance of these findings, it must be pointed out that arginine restriction should result in an increased level of lysine biosynthesis since the enzymes from the corresponding biosynthetic pathway are subject to the general amino acid control system (Jones and Fink, 1982). In the case of the expression of bacterial aminoacyl-tRNA synthetases, it was shown that the level of tRNA aminoacylation is involved in the regulation of the cognate synthetases specific for threonine (Springer et al., 1985) and phenylalanine (Fayat et al., 1983). Assuming that tRNALY” does play a role in the regulation of yeast lysyl-tRNA synthetase, it may be predicted that conditions which promote a decrease in the in uiuo aminoacylation level of tRNALya would allow overexpres- sion of lysyl-tRNA synthetase following activation of KRSl transcription. This possibility will be tested by submitting a lysine leaky auxotrophic strain to lysine starvation. In consid- ering the mechanisms implicated in translational repression, we have to envisage the possibility that lysyl-tRNA synthetase may repress its own synthesis by interacting with KRSl mRNA through its cationic N-terminal moiety. However, an autogenous repression mechanism cannot explain, a priori, the finding that the level of enzyme is not significantly altered following a &fold derepression of its messenger RNA.

Two additional features of the 5’-end of KRSl mRNA are noteworthy. First, this portion of the molecule can be folded into stable secondary structures encompassing nucleotides -50 to +loo. In particular, a four-stem structure, accounting for a total stability of -32.7 kcal, can be generated within the N-terminal part of KRSl mRNA. Second, the amino acid composition of the N-terminal protein sequence encoded by nucleotides +1 to +183 is highly biased, comprising 12 alanine residues in addition to the 13 lysines mentioned earlier (37 alanines and 48 lysines among the 591 residues of the full- length protein). The possibility that the 5‘-end of KRSl mRNA is involved in the repression of lysyl-tRNA synthetase expression via the formation of stable secondary structures and/or its abnormal codon composition is being investigated.

Acknowledgments-We are indebted to J. Rossier and J.-P. Le Caer for the protein sequence analyses, to R. Giege for communicating results prior to publication, to Y. Surdin-Kejan and D. Thomas for fruitful advices, and to M.-T. Latreille for excellent technical assist- ance d.uring part of this work.


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Documents

The Yeast Lysyl-tRNA Synthetase Gene