... Strasbourg Ceden and the BLaboratoire d'Enzyrnologie, Centre National de la ...... 15414 a and P Subunit Genes of Yeast Phenylalanyl-tRNA Synthetase.
Vol. 263, No. 30, Issue of October 25, pp. 15407-15415,1988 Printed in L‘. S A .
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, InC.
Structure andExpression of the Genes Encoding the (X and ,8 Subunits of Yeast Phenylalanyl-tRNA Synthetase* (Received for publication, March 21, 1988)
Ambaliou Sanni$, Marc Mirandes, Jean-Pierre EbelS, YvesBoulangerS, Jean-Pierre Wallerg, and Franco FasioloSq From the SZnstitute de Biologie Mol6culaire et Cellulaire du Centre National de la Recherche Scientifique, Laboratoire de Biochimie, 15, rue Rene Descartes, 67084 Strasbourg Ceden and the BLaboratoire d’Enzyrnologie, Centre National de la Recherche Scientifique, 91190 Gif-sur-Yuette, France
The two genes FRSl and FRS2 encoding, respectively, the large (a) and small (6) subunits of cytoplasmic phenylalanyl-tRNA synthetase from bakers’ yeast have been cloned and sequenced. The derived protein primary structures are confirmed by peptide sequences evenly distributed along the readingframes. These predict a subunit M,of 67,347for a and 57,433 for j3, in good agreement with earlier determinations carried out on the purified protein. These subunit sequences have been compared to those of Escherichia coli phenylalanyl-tRNA synthetase as well as to the small 6 subunit of the corresponding yeast mitochondrial enzyme; limited but significant homology was found between the twoCY subunits on the one hand and between the three @ subunits on the other hand. The results suggest that these three enzymes, from E. coli, yeast cytoplasm, and yeast mitochondria, have strongly diverged from one another. The initiation sites of transcription have been determined for both yeast genes. Their S’-upstrearn regions show no sequence similarities thatwould have indicated a coordinate controlof gene expression at the transcriptional level. Measurements of steady-state levels of FRSmRNAs in overproducing strains indicate that thereis no restriction in mRNA synthesis. Therefore the control of gene expression, leading to a balanced synthesis of a and j3 subunits, is likely to occur at the translational level.
Aminoacyl-tRNA synthetasesconstitute a family of 20 functionally homologous enzymes displaying a large diversity in molecular weights and subunit structures (Schimmel and SOH, 1979). Whereas the majority of aminoacyl-tRNA synthetases is formed of monomers or oligomers made up of identical subunits, phenylalanyl-tRNA synthetase is generally encountered as a tetramer with an azPz type structure. With a few exceptions, homologous aminoacyl-tRNA synthetases from prokaryotes and eukaryotes display the same quaternary structures. This is particularly exemplified in the case of phenylalanyl-tRNA synthetase,which occurs as a tetramerof the a& type in Escherichia coli (Fayat et al., 1974), yeast
mitochondria1 (Diatewa andStahl, 1980) and cytoplasma (Fasiolo et al., 1970),and mammals (Tanaka et al., 1976; Pailliez and Waller, 1984). However, the molecular weights for the large ( a )and small (0)subunits of E. coli phenylalanyltRNAsynthetase (87,000 and 37,000 (Fayat et al., 1983; Mechulam et al., 1985)) differ remarkably from those determined for the corresponding enzyme from yeast mitochondria (72,000 and 57,000 (Diatewa and Stahl, 1980)) or cytoplasrna (74,000 and 63,000 (Fasiolo et al., 1970)) and from sheep liver (71,000 and 63,000 (Pailliez and Waller, 1984)). The genes coding for the a ( P h e T ) and p(PheS) subunits of the E. coli enzyme were isolated (Hennecke et al., 1977) and their structure resolved (Fayate et al., 1983; Mechulam et al., 1985). In addition, the gene MSFl encoding the small subunit (p) of yeast mitochondrial phenylalanyl-tRNA synthetase was recently isolated and characterized (Koerner et al., 1987). It encodes a polypeptide of M, 55,000 displaying significant homologies with the @ subunit of the E. coli enzyme. The availability of cloned genes for the a and @ subunits of the yeast cytoplasmic enzyme would offerthe possibility to study the structuralrelationship, if any, with the E. coli enzyme. Recently, the cloning of a portion of the genes coding for the a (FRSI ) and (FRSZ)’ subunits of the yeast cytoplasmic phenylalanyl-tRNA synthetase was accomplished by probing a Xgtll recombinant DNA library with antibodies directed to the purified enzyme (Mirande et ai., 1986). In the present work, we have used these DNA probes to clone the complete genes encoding the two subunits, together with their promoter regions. Their nucleotide sequences were determined. Comparison of the deduced amino acid sequences with those of the a and @ subunits of E. coli and the @ subunit of yeast mitochondrial phenylalanyl-tRNA synthetase clearly establishes structural relationships for their large and small subunits, respectively. However, the extentof homology israther low, in line with the large differences in subunit molecular weights between E. coli and yeast phenylalanyl-tRNA synthetases. The transcription initiation sites of FRSl and FRS2 genes were also determined. No sequence similarities were found in the 5‘ upstream regions of the two genes. Hence the mechanism of coordinate control of their expression does not appear to involve a common regulatory sequence. MATERIALS AND METHODS
* This work was supported by grants from the Centre National de la Recherche Scientifique. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paperhas been submitted to the GenBankTM/EMBL Data Bank with accession number(s) 503964 and 503965. V T o whom correspondence should be addressed.
Yeast, Bacteria, Plasmids,Gene Libraries, and Growth Media-The yeast genomic bank from Saccharomyces cereuisiae strain FLlOO in The abbreviations used were: FRSI and FRSZ designate the two genes encoding the respective a and @ subunits of cytoplasmic phenylalanyl-tRNA synthetase from bakers’ yeast; Pipes, l,4-piperazineethanesulfonic acid; kb, kilobases; bp, base pairs; SDS, sodium dodecyl sulfate.
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Subunit Genes of Yeast Phenylalanyl-tRNA Synthetase
the plasmid vector pFLl was a gift from Dr. F. Lacroute (IBMC, tions, fractionation of tryptic digests, analysis, and sequencing of Strasbourg, France), This bank was constructed by integrating apool purified peptides were carried out as published (Robbe-Saul et al., of Sau3A fragments into the plasmid vector. We also used the yeast 1977; Potier et al., 1980). genomic bank (partial Sau3A digest) in the plasmid vector pJDB207 kindly provided by Dr. B. J. Beggs (University of Edinburgh, United RESULTS Kingdom). The recipients for yeast transformation were strains Ura3-, Leu2-, and the double mutant Ura3-, LeuP-, all isogenic to Isolation of Complete Genomic Sequences Encoding the a FL100. Parental and transformed yeast strains were grown on YNB and p Subunits of Yeast CytoplasmicPhenylalanyl-tRNA Syn(0.67% yeast nitrogen base without amino acids, 2% glucose, w/v). thetase-The fact that isolated a orsubunitare inactive Transformations of yeast andE. coli and preparation of nucleic acids and that only the tetrameric molecule a& is active (Fasiolo were done using standard procedures. Enzymes and Reagents-Restriction endonucleases, T4 DNA li- et al., 1975) imposes a precise strategy of cloning. The FRSl gase, and E. coli DNA polymerase I (Klenow fragment) were pur- and FRSB genes should be cloned into two plasmid vectors chased from Boehringer Mannheim, West Germany. [(u-~'P]~ATP, which have different auxotrophic markers so that cotransfora-35S-dATP,and '251-proteinA were purchased from Du Pont-New mation of a double recipient mutant can be checked by transEngland Nuclear. formation to prototrophy. Hybridization Procedures-DNA probes from recombinant Xgtll Recombinant Xgtll clones encoding a portion of the a and or recombinant plasmids were purified bygel electrophoresis or p subunits of yeast cytoplasmic phenylalanyl-tRNA synthesucrose gradient centrifugation. They were labeled by nick translation as described by Maniatis et al. (1982). DNA probes cloned in M13 tase were previously obtained by probing a h g t l l recombinant phage were labeled by chain extension using the Klenow fragment of DNA expression library with antibodies directed against the E. coli DNA polymerase I and [ c ~ - ~ ' P ] ~ A T P . yeast T h e genomic bank purified enzyme (Mirande etal., 1986).To obtain the complete in vectors pFLl or pJDB 207 was screened by the high density colony nucleotide sequences corresponding to thetwo genes,we have screening procedure described by Hanahan and Meselson (1983). used as DNA probes the 1400-bp-longEcoRI-EcoRI fragment Positive clones were purified by two additional cycles of screening. Southern and Northern blot hybridizations were carried out according from XPhell and the220-bp-long EcoRI-XhoI fragment from XPhe6, corresponding to the a and p subunit of yeast phento the procedures described by Maniatis et al. (1982). Mapping of FRSI- and FRS2-mRNA Termini-S1 nuclease pro- ylalanyl-tRNA synthetase, respectively. tection experiments were performed essentially as described by Berk By probing the DNA library constructed in the pFL1vector and Sharp (1977). Synthetic oligonucleotides were used as primers with the DNA fragment coding for the large subunit (a,M, for chain extension of the appropriate single-stranded DNA templates 74,000) of phenylalanyl-tRNA synthetase, the FRSlgene was in M13 containing the 5"upstream region of FRSl and FRS2 and 6-kbp AccI part of the coding regions. The resulting double-stranded M13 vector isolated on a 10-kbp-long DNA insertanda was cleaved a t the unique KpnZ site (at position -130) in the 5'- fragment (Fig. la). Following digestion with EcoRI and StuI, upstream region of FRSI, and thelabeled single strand was isolated large fragments were cloned into bacteriophage M13mp8 or directly on an acrylamide-urea sequencing gel. Chain-elongated DNA M13mp9 for DNA sequencing. primer, complementary to theFRS2 coding region, was digested with The stop codon of FRSB was within the yeast DNA insert AccI (coordinate -291). The isolated single strands (5000 cpm Cer- of the isolated XPhe6 recombinant. We took advantage of the enkov) were hybridized to 15 pgof poly(A+) mRNA prepared from presence of a unique BstEII site in the DNA insert to clone wild type yeast cells. After incubation for 8 h at 42 "C in 10 mM Pipes buffer, pH 6.5, containing 400 mM NaCl and 50% formamide (v/v), the remaining of the coding sequence and the 5'-upstream the hybridization mixture was diluted 10-fold in S1 buffer containing region. We observed by Southern mapping of genomic DNA various amounts of S1 nuclease and digested for 2 h at 25 "C. S1- that these regions are within a 1.6-kb BstEII fragment. We resistant fragments were separated on a 6% sequencing gel and could characterize this fragment together with the rest of the visualized by autoradiography. The unlabeled oligonucleotides were gene on a clone obtained by hybridization of the genomic used to generate T, C, G, A tracks of the DNA region encompassing bankin the pJDB207 vector (having Leu2 as selectable the startsof transcription. The position of the S1-resistant fragment marker) with the 220-bp EcoRI-XhoI fragment (see Fig. l b ) . indicates the site of transcription initiation. Nucleotide Sequences of the FRSl and FRS2 Genes-The Northern Analysis-Poly(A+) mRNA (10 pg)was subjected to formaldehyde agarose gel electrophoresis as specified in the Gene- strategy of Dale et al. (1985) was used to generate a set of Screen manual. RNA was transferred to nitrocellulose as described overlapping clones (Fig. 1, a and 6). The complete nucleotide by Thomas (1980). Blots were probed either with the synthetic sequences of FRSl (Fig. 2) and FRS2 (Fig. 3) genes were oligonucleotides used in S1 mapping experiments or with large DNA determined on both strands. Long open reading frames of fragments homologous to the coding sequences; these large DNA fragments were generated by chain extension with Klenow polymer- 1785 and 1509 bp were found for the genes encoding the a and p subunits of phenylalanyl-tRNAsynthetase, respecase. Western Blot-Protein samples were run on 10% polyacrylamide tively. The predicted protein encoded by FRSl is composed gels in the presence of 0.1% SDS (Laemmli, 1970).Conditions for the of 595 amino acid residues, accounting for a M, of 67,347, and transfer of proteins to nitrocellulose membranes were as described in that encoded by FRS2 consists of 503 amino acids with a the Schleicher & Schuell manual number 2. The protein correspond- calculated M, of 57,433. These values are significantly lower ing to phenylalanyl-tRNA synthetase was detected as described above, using DEAE-cellulose-purified antibodies raised against the than those previously determined for the a ( M , 74,000) and @ native enzyme (5-10 mg/ml) and "'I-protein A (0.1 mCi/ml). Protein (Mr 63,000) subunits of the purified enzyme (Fasiolo et al., 1974), by SDS-polyacrylamide gel electrophoresis according concentration was estimated according to Bradford (1976). DNA Sequence Analysis-Fragments (1.4-1.6 kb) of FRSl and to Laemmli (1970), but are in good agreement with earlier FRS2 were cloned into Ml3mp8 and M13mp9 vectors (Vieira and determinations (Fasiolo et al., 1970) performed at neutral pH Messing, 1982). Overlapping clones of each strand were obtained in phosphate buffer (63,000 and 56,000, respectively, for the using the cloning strategy described by Dale et al. (1985) with the following modification: digestion by Hind111 was carried out at37 "C a and p subunits). Additional evidence excludes that such after hybridization at 68 "C. Nucleotide sequences were determined discrepancies might be due to theisolation of truncated genes; using the dideoxynucleotidechain termination method (Sanger et al., genetic maps of FRSl and FRS2 were verified by genomic 1977). Southern analysis; besides in the case of FRSl thesize of the acid se- gene-encoded mRNA corresponds to that of mature FRSlComputerAnalysis of AminoAcidSequences-Amino quences were analyzed with programs from the University of Wiscon- mRNA (see below). Unusual mobility on SDS-polyacrylamide sin Genetics Computer Group edited by J. Dereveux and P. Haeherli: "Best fit and Gap" to align two sequences, "Dotplot" and "Pepplot" gel has been observed for c-fos and adenovirus E1A proteins which are proline-rich (Van Beveren et al., 1983; Ferguson et to visualize the homology between two sequences. Protein Chemistry-Separation of a and p subunits, tryptic diges- al., 1984).For these proteins this high proline content may
Phenylalanyl-tRNA Synthetase
15409
mRNA and at positions -36, -41, -58, and -59 for J'RS.2mRNA. The presence of undigested probe, even at high level of S1 nuclease (Fig. 4, A and B, lane 3 ) might suggest that there is an upstream promoter for FRSl and FRS.2 genes. However, we do also observe the presence of undigested material in the control lane where no RNA was added, indicating that our single-stranded DNA probe is contaminated by double-stranded material, having the labeled 5' end protected from nuclease attack by the corresponding complementary sequences of the opposite strand. This form might be generated by mechanical shearing of the circular DNA template (see the method of preparation of the labeled singlestranded DNA probe). While TA-rich sequences are located between nucleotides -82 to -44 €OT FRS.2, no such sequences were found in the 5"upstream region of F R S l . In addition, the 5"upstream regions of both genes display no significant homologies. Assignment of the FRSl and FRS2 Gene Products to Yeast Cytoplasmic Phenylalanyl-tRNA Synthetase-FRSI and FRSS gene products were previously identified, by immunochemical approaches (Mirande et al., 1986), as the a and /3 1 kb subunits of yeast cytoplasmic phenylalanyl-tRNA synthetase, respectively. Additional evidence supporting these assignments is brought by the examination of the amino acid contents of the predicted polypeptides encoded by these two genes.The amino of yeast phenylacid compositions of the a and subunits alanyl-tRNA synthetase previously determined on the purified enzyme (Robbe-Saul et al., 1977) are in good agreement with those deduced from the FRSl and FRS2 reading frames (Table I). In addition, an N-terminal proline residue was c reported for the a subunit, whereas a blocked N-terminal residue was expected for the p subunit (Robbe-Saul et al., 1977). These dataare in accordance with the N-terminal amino acid sequences predicted for the FRSI and FRSB gene products, respectively: Met-Pro-. . . and Met-Ser-. . . Indeed, .%... *.** taking into account the observed patterns of the effect of the I 0J( I C BalEII EcoRl BsIEIl N d e l second amino acid on post-translational modifications in yeast (Huang et al., 1987), the N-terminal methionine is c"-l generally removed when the second amino acid is a proline, lkb FIG. 1. Restriction map of the FRS genomic regions and the latter remaining unblocked, whereas methionine is resequencing strategy. a, FRSl gene; b; FRS2 gene. The restriction moved and serine is blocked when the 2nd residue is a serine. N-terminal residue maps were determined by Southern analysis using yeast genomic Therefore, N-acetyl-Ser is likely to be the DNA. The FRSl and FRS2 coding sequences are indicated by a heauy of mature ,8 as was already found for cytoplasmic methionylarrow. The boxes represent the length of the DNA probes isolated and valyl-tRNA synthetases from yeast (Fasiolo et al., 1985; from h g t l l recombinants and used to clone the complete genes. Chatton et al., 1988). Arrows indicate the extent of sequence obtained from each of the Moreover, the amino acid compositions and/or amino acid cloned fragments. The position of FRSl and FRS2 genes in the recombinant plasmid is shown above the genomic map for FRSl and sequences surrounding 7 cysteine residues in the a subunit under it for FRS2. Dashed lines indicate the region sequenced relative and the unique cysteine residue of the subunit of phenylto the insert. alanyl-tRNAsynthetase were reported (Robbe-Saul et al., 1977; Potier et al., 1980). The corresponding sequences are result in an unusual structure which is revealed by the elec- present in the predicted proteins encoded in the FRSl or trophoretic behavior on SDS-polyacrylamide gels. This can- FRS2 genes (Figs. 2 and 3). Besides, the amino acid componot be the explanation to the size discrepancy between the sitions of several pure trypticpeptides were obtained from the experimental values and those deduced from the gene se- a subunit. The corresponding sequences are underlined in quence of yeast phenylalanyl-tRNA synthetase subunitssince Fig. 2. Altogether tryptic peptides of a (including those contheir proline content is not atypical. The differences might taining cysteine) cover 306residues out of 595 (51%)between be due to thedifferent buffer systems used for SDS-polyacryl- positions 46 and 556. Similarly short tryptic peptides of p could be sequenced and aligned along the translatedsequence amide gels. The codon bias indexes of 0.42 ( F R S l ) and 0.44 (FRSS), as shown in Fig. 3. They account for 134 residues out of 503 calculated as described by Bennetzen and Hall (1982), are (27%) and arelocated between positions 42 and 483. Furthermore, the a and /3 subunits of the E. coli enzyme characteristic of proteins of medium to low abundance. The mRNA initiation siteswere determined by nuclease S1 display some sequence similarities with the amino acid seprotection experiments, as shown in Fig. 4. The results indi- quences deduced from FRSl and FRSB genes (see below). cate initiation sitesat positions -15, -27, -32, -40, and -41 Taken together, these results provide compelling evidence upstream from the translationinitiation codon for F R S l - in favor of the isolation of the two genes codingfor the a and
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15410
a and B Subunit Genes of Yeast Phenylalanyl-tRNA Synthetase -360
CACACTGATT~TTTATTAGTTCTTTTMGTGCCCMCTT~CAG-G-CACCCC~~TAC~AGTGTATGTM~~~CTTGM
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FRSI and translated amino acid sequence of the a subunit. The region corresponding to the major and minor mRNA 5' endsis ouerlined. The open reading frame spans from nucleotide 1 (A of the initiating ATG) to 1785, after which a stop codon (TAA) occurs. The one-letter symbol of each amino acid is printed under the first letter of the corresponding codon. Trypticpeptides that gave an unambiguous amino acid composition (or sequence) are underlined.
-91
T T C M T G T A T T T C T C ~ G C C T ~ ~ ~ A G ~ U T C ~ T G T T C ~ T A G G G C A G M T A G C A T M C A C A C A T C M T M C A C C U G C C A-1G C
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TTUCGGTTGAGCCTCTA~TTGTCTCTWCACMT~CCMTCCCGTTT~~~CGCC-CTTCMC-TAGMTTATGGATGTCTC;
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680 for the E. coli enzyme, which are aligned on a diagonal N- andC-terminal tase. To further document the assignment of the FRSl and (Fig. 5A). Thisalignmentpointsout FRSP genes to the CY and p subunits of the yeast enzyme, the extensions for the E. coli CY subunit, as compared to the yeast cotransformation of yeast cells with high copy number plas- CY subunit (Fig. 5 C ) . Concerning p subunits, more extensive mids bearing the two genes was undertaken. Elevated levels correlations were found (Figs. 5B and 6). Provided thata gap E. coli enzyme, large of phenylalanyl-tRNA synthetasecould not beobserved under of about 40 residues be introduced in the theseconditions due tothe failure to overproduce the p homologous regions encompassing residues 220-280 and 320480 for the yeast enzyme, and residues 100 to 310 for the E. subunit (see below). Homology of Yeast Cytoplasmic Phenylalanyl-tRNA Synthe-coli counterpart, canbe visualized. These sequence similarities tase to the Corresponding Enzymes from Yeast Mitochondria extend over the C-terminalregions of both enzymes, pointing of about 100 amino acid residues and E. coli-The FRSl and FRS2 gene products were com- out an N-terminal extension pared to the (Y and p subunits of E. coli phenylalanyl-tRNA for the p subunit of yeast phenylalanyl-tRNA synthetase(Fig. 5C). Dot matrix comparison of a (yeast) versus /3 (E. coli) or synthetase (Fig. 5 ) . For a subunits, dot matrix comparison reveals four small stretchesof homology between residues 310 p (yeast ) versus a (E. coli) displayed no significant homoloand 580 of the yeast enzyme and between residues 420 and gies.
a and
Subunit Genes of Yeast Phenylalanyl-tRNA Synthetase -331
I~"~GCCA~~A~TTTTTCTGTTTTTCTCTACCTCTTTT~TTTTCAGT~CTTTGTTC;TCTCTACG - 1T 8 1
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ATGTCCAMCTAffiCCCTCMGTTffiTMffiTCffiTCAGGCTA~GCTTTC~GMCGGCTGGATCGCC~CGCCTC~CGAGCTT ~
~
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CM~TGTTCTTTCCCCTTTGMCTCTCTTT~GCCCACMCMGTTAGAGTTTTCCMGGTCGACACGGTTACGTATGACTTGACCAM Q D V L S A L N S L K A H N K L E F S K V D T V T Y D L T K
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; T G T C T - C T T C C M T T A - T T C T - - C T A ~ T ~ T T C U
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GMCTCTCTCCGCIAMTTGCIIAMTACCGATTT~TGAGCTTACT~TG~CGCMTCTATTCTAGCGCAMTCMGMCTCMCTCGCAT 4 5 0 E L S A K L Q N T D L N E L T D E T Q S I L I P I r N N S H
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CT~~AGCATTGACCCCMGATTTTGMCTC~CTT~GIIAM~GTTMTTGCTC~GGT~TCACA~TTTCAGTGT S 4C0A C C ~
L
are as describedfor Fig. 2. The open reading frame corresponds to nucleotides 1-1509. Sequenced tryptic peptides are underlined.
CTGACCCGGCTTTGGTATCGTTTTATCACGGCTT~ACCCGGTCGACCATATTGMTC~C - 2 7 1
-270
-90
FIG.3. Nucleotide sequence of FRS2 and translated amino acid sequence of the fi subunit. The symbols
15411
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AMGCCGTT;ACGMCTCAGCGGAGATTCCG~~TCCATCGGTTATCGTTACMCTGGMGCCAGMGMTGT~~TTGGTCTT~GMCTCT 990 -K
991
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M G C C T T A C M T T T C M T T C T C M f f i T G T G C ~ T A T C T T C A f f i T G C T C T T C A C C C C T T ~ C A M G T C A ~ ~ G G M C T T T T A ~ C ~1 T 20 T
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GM~CUGAGTTCTC~CCWICCTUCC-TT~GAMCC~ATCTTACCTCC~CAT~~~TCTCCACCMTGCATACM~CTTGMCTGTT; G
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CACTCCACAGCCATCTCTGCCAGMCTTGCTGCACGAT1TGTTGTTTTCTATCGACCGTGTTTTCCGT
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t o ~ l MCGMGCAGTTGACGCCACCCATTTGGCC~AATTCCACCAGGTGG-G~TGTTCTTGCCGACTACMCATTACTCTGGGTGACCTGAT; N 1171
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A T C T T T T C T T G G C A C G M C T G C T T T G C - T G ~ ~ ~ T C C G T C C M R T T C T A1350 I
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MGTTCAT-~GTTTTTC~GMCTTGGGTGTCACCGGTTTGA~TTCMGCCTAC~TACMTCCTTACACC~GCCATCMTG~ K
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GtTCAT~GTCTCTTTGCACTTTATCG~CCMTCCTGCTGCTA~TTGGACGMCTGACTTGTACGMCTT~GCTCAGCAGTTCCTCTAG G H K V S L D F I E T N P A A R L D E D L Y E .
1530
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TTTTATATCGTCTGTCCCCMTTTTGTAGTAGTTATTCTACACIIAMC~TATATACGTATATGTTMTATAG 1620
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In addition, when the MSFl gene product, corresponding to thesmall ((3) subunit of yeast mitochondrial phenylalanyltRNA synthetase, was compared to the two subunits of the corresponding enzymes from E. coli and yeast cytoplasma, a good correlation between the three /3 subunits could be observed (Fig. 6). A stronger homology was found between the @ subunits of E. coli and yeast mitochondria, than between yeast mitochondrial andcytoplasmic /3 subunits. Northern Analysis-The yeast recipient strain produces mature FRSl- and FRS2-mRNAs whose sizes correspond to 2 and 1.7 kb, respectively (Fig. 7A and B, lanes 1), in accordance with the sizes of the coding regions determined previously. In yeast transformants bearing the FRSl gene on a high copy numberplasmid,therelativeconcentration of FRSl-mRNA was increased by a factor of 25 as judged by scanning measurements(Fig. 7A, lane 2). Analysis of a protein crude extract by the protein blotting procedure showed that these yeast transformantsoverproduce a truncated CY subunit
1674
is degraded by of M, 46,000 indicatingthatthissubunit proteases when unassociated to the @ subunit. It must be stressed that trace amounts of a protein band of similar M , could be detected in samples of purified phenylalanyl-tRNA synthetase due to uncontrolled proteolysis (Fig. 7C, lane 3 ) . On the other hand, when the recipient strain was transformed withtheplasmid-borneFRS2 gene, no overproduction of FRS2-mRNA of the appropriate size was observed (Fig. 7B, lane 2). Insteada long transcript of 4.8 kb could be detected on theblot (Fig. 7B, lane 2); it displayed an intensity approximately five times as high as thatof normal FRS2-mRNA. A Western blot performed on the corresponding crude extract did not reveal any overproduction of the (3 subunit (data not shown). DISCUSSION
Phenylalanyl-tRNA synthetase from E. coli and yeast displaythesame oligomeric structure of the a&2 type. The
15412
(Y
and p Subunit Genes of Yeast Phenylalanyl-tRNA Synthetase
B
A
T C G A l 23
TABLE I Amino acid composition of yeast cytoplosmicphenylalanyl-tRNA synthetase subunits For protein analyses, numbers were calculated on the basis of estimated M, (70,000 for (Y and 60,000 for 8) and rounded to the nearest integer. a B Amino acid ProteinDNAProteinDNA
t
Ala 27 Arg Asn ASP Asx 9Cys" Gln Glu Glx 34 GlY His Ile Leu 47 LYS Met" 30 Phe 30 Pro Ser 30 Thr Trp* Val 34
ZtJ
-32 * - 27 e -15
FIG. 4. Determinati.on of the 5' ends of FRSl-mRNA ( A ) and FRS2-mRNA ( B )transcripts. Lane 1 shows a control reaction without mRNA (6000 units of S1 nuclease added). Lanes 2 and 3 show digestions of RNA-DNA hybrids by 2000 and 6000 units of S1 nuclease (Boehringer Mannheim), respectively. The SI-protected bands are displayed along with TCGA sequencing reactions of the coding strand, using the respective phosphorylated synthetic oligonucleotides as primers. The coordinates of the oligonucleotides were +44 to +61 for FRS2-mRNA and +71 to +87 for FRSI-mRNA. The major transcription sites are indicated by arrows.
primary structures of the a and B subunits of the E. coli enzyme were previously resolved via cloning and sequencing of the corresponding genes, PheS and PheT, respectively (Fayat et al., 1983; Mechulam et al., 1985). The deduced molecular weight of the tetramericenzyme is about 248,000. In thepresent work we report theisolation and sequencing of the FRSl and FRS2 genes encoding the a and /3 subunits of yeast cytoplasmic phenylalanyl-tRNA synthetase. The corresponding molecular weight of the native enzyme (Mr = 249,500) is very similar to that of the homologous enzyme from E. coli. However, individual subunits exhibit very dissimilar sizes: 87,000 and 67,500 for the a subunits from E. coli and yeast; 37,000 and 57,500 for the corresponding B subunits. Moreover, the dot matrix profiles shown in Figs. 5 and 6 reveal nostrong homology between individual subunits. Taken together, these results indicatethat thetwo structures have strongly diverged. In addition, as a better homology was found between the p subunits of E. coli and yeast mitochondria (homology score of -164) and E. coli and yeast cytoplasma (-144), than between the cytoplasmic and mitochondrial B subunits from yeast (-72) (Fig. 6), it may be speculated that the mitochondrial and cytoplasmic enzymes from yeast have evolved independently from a common prokaryotic type ancestor. A tentative alignmentof the sequences of the corresponding
37 25 31 42
41
28 20
27 19 25 34
62 2
73 9 18 47
1 25 39
70 30 13 27 62 40 12 28 22 29 29 16 25
77 27 15 43 55 46 13 29 30 38 30 4 39
28 12 27 58 41 14 26 20 32 30 15 24
14 36 60 10 39 20
595 Total616 522 503 Cys and Met were determined as cysteic acid and methionine sulfone, respectively, after performic oxidation of the protein. * Trp was estimated after hydrolysis with methane sulfonic acid. The translated sequence includes the initiating Met absent in mature subunits.
t C
B
1
280 317
116
B
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1
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FIG. 5. Homology between a and fl subunits of yeast (onlinate) and E. coli (abscissa)phenylalanyl-tRNA synthetases. The comparison uses the Dot Matrix program from the University of Wisconsin Genetics Computer Group.Average score values were calculated for pairs of 25 amino acid segments, using the mutation matrix of Staden (1982). If the average score value was equal to or greater than 25, a dot was printed at the corresponding position in the matrix. A and B represent the homology between the two a and the two 8 subunits, respectively, and C, the alignment of the corresponding sequences.
subunits from yeast and E. coli, based on thelimited sequence homologies found, is schematized in Fig. 5C. The B subunit of the yeast enzyme shows an N-terminal chain extension of about 120 residues, whereas alignment of the two a subunits
a and /3 Subunit Genes of Yeast Phenylalanyl-tRNA Synthetase
15413
Y e a s t Cyto Y e a s t Cyto
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Y e a s t Mito
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Y e a s t Mito
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Y e a s t Mito
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c F R S 1 F RS2-m
w -30
FIG.7. Expression of FRSl and FRS2 genes. A and B, Northern Blot of FRSl ( A ) - and FRS2 (B)-mRNAs. Lane I , recipient strain; lane 2, yeast transformant bearing FRSl ( A ) or FRS2 ( B ) genes on a high copy number plasmid. The positions of FRSI- and FRS2-mRNAs are indicated by arrows: there respective sizes were estimated to 2 and 1.7 kb. The VAS gene (3.2 kb) was used as an internal probe. This gene codes for yeastvalyl-tRNA synthetase (Jordana et al., 1987). C, Western blot of the transformant bearing the FRSI gene on a high copy number plasmid lane I , recipient strain transformed with the cloned FRSl gene (50 pg of crude protein extract); lane 2, recipient strain (50 pg of crude protein extract); lane 3, purified yeast phenylalanyl-tRNA synthetase (0.2 pg); lane 4 contains markers of known M, (xIO-'). The overproduction of the a subunit was visualized using antibodies raised against either the native enzyme or the purified Q subunit.
moiety (residues 1-120) of the j3 subunit of yeast phenylalanyl-tRNA synthetaseto residues 1-100 or 700-795 (or even 300 Y e a s t Mito 100-700) of the a subunit of the E. coli enzyme revealed no E.wli 261 significant homology. From the functional point of view, it is noteworthy that 423 Y e a s t Cyto residues Lys-2, Lys-61 and Lys-106 from the E. coli a subunit, which were found to be implicated in the bindingof the CCA 351 Y e a s t Mito arm of tRNA (Hountondji et al., 1987), belong to the NE.mli 312 terminal part of the polypeptide chain for which no homoloYeast Cyto 476 gous region is revealed in the yeast enzyme (Fig. 5C). Moreover, affinitylabeling experiments of yeast phenylalanyl409 Yeast Mito tRNA synthetase with 3"oxidized tRNAPhe (Renaud et al., 1982) have shown that the CCA arm of tRNA reacts with E.wli 327 Lys-325proximal to the uniquecysteineresidue of the B Yeast Cyto 503 subunit (Fig. 3). Furthermore, thebody of tRNAPhewas shown to interactexclusively with the large a subunit in thecase of Yeast Mito 467 the E. coli enzyme (Khodyrevaet al., 1985) and with the small /3 subunit in the case of the yeast counterpart (Baltzinger et 474 Y e a s t Mito al., 1979). These results further exemplify the strong diverFIG.6. Protein sequence homologies between E. coli, yeast gence displayed by these two enzymes. mitochondrial and yeast cytoplasmic B subunits of phenylaAnother striking featureis the presence in the a subunit of lanyl-tRNA synthetase. Alignment of the three polypeptides is yeast phenylalanyl-tRNAsynthetase of a pentapeptide based on the Kanehisa alignment program (Kanehisa et al., 1984) by weighing with the mutation data matrix (Dayoff et al., 1983). Con- KLSKP (positions 86-90) which closely resembles the conservative changes are marked bydashes, identical residues are boxed. sensus sequence KMSKS which was proposed to be involved The related homology scores are: -164 for E. coli versus yeast mito- in theCCA binding of tRNA (Hountondjiet al., 1986a). Thus, chondrial; -144 for E. coli versus yeast cytoplasmic -72 for mito- it may be speculated that, in the native a 2 p 2 structure of yeast chondrial versus cytoplasmic yeast 0 subunits. phenylalanyl-tRNA synthetase, the N-terminalregion of the a subunit, bearing the KLSKP sequence, is in the vicinity of points out N- and C-terminal extensionsof about 100 amino Lys-325 of the /3 subunit, leading to the formation of the acids in the case of the prokaryotic polypeptide chain. In E. active site of the enzyme. The finding that no lysine residues coli the PheS and PheT genes are adjacent and transcribed from the CY subunit of the yeast enzyme were labeled with from a single promoter, with an intercistronic region com- tRNAPheox (Renaud et al., 1982) may be explained by the prising 14nucleotides (Fayat et al., 1983). Taking into account flexibility of the CCA arm which was shown to interact with the striking similarity inmolecular weights displayed by the several lysine residues in the case of E. coli phenylalanyltetrameric enzymes from E. coli and yeast cytoplasma, we tRNA synthetase (Hountondjiet al., 1985; Hountonjdi et al., considered the possibility that the a and /3 subunits of yeast 1986b). Therefore,the labeled lysines arenot necessarily phenylalanyl-tRNA synthetase may have arisen through in- involved in catalysis but should be clustered near the active tramolecular rearrangements of the a and p subunits of the site. prokaryotic enzyme. However, comparison of the N-terminal The involvement in the tRNA aminoacylation reaction of
15414
a and
P Subunit Genes of Yeast Phenylalanyl-tRNA Synthetase
the contact area of the two subunits is further strengthened by the following observations: (i) isolated subunits from the yeast enzyme (Fasiolo et al., 1975), as well as from the E. coli enzyme (Ducruix et al.,1983) are unable to catalyze the ATPPPi exchange and the tRNA aminoacylation reactions; (ii) whereas tRNA and ATP were found to interact exclusively with the p subunit of yeast phenylalanyl-tRNA synthetase, phenylalanine could be cross-linked to both subunits (Baltzinger et al., 1979). It is also noteworthy that a QIGH sequence, closely related to the sequence HIGH that is believed to correspond to the ATP-binding region (Schimmel, 1987), is located at the Cterminal extremity of the a subunit from yeast phenylalanyltRNA synthetase (positions 563-566). If this sequence is relevant to ATP binding, this would imply that in the threedimensional structure of the enzyme, the C-terminal region of the a subunit lies close to itsN-terminal region. The involvement of N- and C-terminal regions of E. coli methionyl-tRNA synthetase in catalysis, via the folding of the Cterminal backbone back toward the N-terminal domain, was recently reported (Brunie et al., 1987). To our knowledge, yeast mRNAs are always encountered as monocistronic translation units. S1 mapping experiments clearly indicate that FRSl and FRS2 genes are transcribed independently. This situation differs from that found for the E. coli enzyme. In that case, the two genes coding for the a and p subunitsare organized inan operon PheS, T. In addition, it was shown that the expression of E. coli phenylalanyl-tRNA synthetase is under the control of a phenylalanine-mediated attenuationmechanism. In yeast, where transcription andtranslationare disconnected, it seems very improbable that an attenuationmechanism may coordinately control the level of expression of the two subunits of phenylalanyl-tRNA synthetase. This assumption is confirmed by the analysis of the 5”upstream regions of both genes. No short open reading frame coding for a phenylalanine-rich peptide and no characteristic stem-loop structures were found. Moreover, no highlyconserved sequences were found between the FRSl and FRSl 5”upstream regions, suggesting that these two genes are regulated independently. Our results on the expression of FRSl gene suggest that regulation may occur at thetranslational level. Measurements of steady state levels of FRSl-mRNA show that yeast cells containing the FRSl gene on a high copy number plasmid (pFL1) overproduce at least 25 times as much FRSl -mRNA as control cells, a value which probably reflects the increase in gene dosage. This supports the idea that there is no restriction of mRNA production at the transcriptional level. From the Western analysis shown in Fig.7C, it could be estimated that the amount of a subunit in the plasmid containing cells is only 10 times as high as in control cells. However, it is difficult to conclude from these analyses that theyeast cells use a translational control mechanism to compensate for excess copies of this FRSl gene, because the native a subunit is subjected to extensive proteolysis in vivo (or during sample preparation). We could not observe any overproduction of mature FRS2mRNA in yeast cells transformed by the autonomously replicating plasmid pJDB207. Instead we found a 5-fold overproduction of a 4.8-kb-long transcript homologous to the FRS2 gene. This is most probably because this gene is transcribed from plasmid sequences, although the 5’ noncoding region of the FRSl gene contains the normal sequences required for accurate initiation of transcription as demonstrated by our S1 mapping analyses with poly(A+) mRNA from wild type cells. Unexpected transcripts, giving rise to altered gene
expression, were also found in the case of a yeast centromere plasmid (Marczynski and Jaehning, 1985). Thus the production of this unanticipated transcript may explain the lack of overproduction of the /3 subunit and hence the absence of increased levels of the tetrameric a& phenylalanyl-tRNA synthetase in yeast cells cotransformed with FRSl and FRS2 plasmid-borne guests. The elucidation of the nature of the regulatory mechanisms leading to coordinate synthesis of the a and p chains will be the object of future studies. Acknowledgments-The skillful technical assistance of G . Nussbaum is gratefully acknowledged. We thank Dr. P. Remy for his gift of antibodies raised against the purified a subunit of yeast phenylalanyl-tRNA synthetase. REFERENCES Baltzinger, M., Fasiolo, F., and Remy, P. (1987) Eur. J.Biochem. 9 7 , 481-494 Bannetzen, J. L., and Hall, B. D. (1982) J. Biol. Chem. 2 5 7 , 30183025 Berk, A. J., and Sharp, N. (1977) Cell 12,721-732 Bradford, M. M. (1976) Anal. Biochem. 72,248-254 Brunie, S., Mellot, P., Zelwer,C., Risler, J. L., Blanquet, S., and Fayat, G. (1987) J. Mol. Graphics 5 , 18-21 Chatton, B., Walter, Ph., Ebel, J. P., Lacroute, F., and Fasiolo, F. (1988) J. Biol. Chem. 263,52-57 Dale, R. M. K., McClure, B. A., and Houchins, J. P. (1985) Plasmid 13,31-40 Dayhoff, M. O., Barker, W.C., and Hunt, L. T. (1983) Methods Enzymol. 91,524-545 Diatewa, M., andStahl, A. J. C. (1980) Biochem. Biophys. Res. Commun. 94,189-198 Ducruix, A., Hounwanou, N., Reinbolt, J., Boulanger, Y., and Blanquet, S. (1983) Biochim. Biophys. Acta 741, 244-250 Fasiolo, F., Befort, N., Boulanger, Y., and Ebel, J.-P. (1970) Biochem. Biophy~.Acta 217,305-318 Fasiolo, F.,Remy, P., Pouyet, J., and Ebel, J. P. (1974) Eur. J. Biochem. 50,227-236 Fasiolo, F., Boulanger, Y., and Ebel, J. P. (1975) Eur. J. Biochem. 53,487-492 Fasiolo, F., Gibson, B. W., Walter, P., Chatton, B., Biemann, K., and Boulanger, Y. (1985) J. Biol. Chem. 2 6 0 , 15571-15576 Fayat, G., Elanquet, S., Dessen, P., Batelier, G., and Waller, J. P. (1974) Biochirnie 5 6 , 35-41 Fayat, G., Mayaux, J. F., Sacerdot, C., Fromant, M., Springer, M., Grunberg-Manago, M., and Blanquet, S. (1983) J. Mol. Biol. 1 7 1 , 239-261 Ferguson, B., Jones, N., Richter, J., and Rosenberg, M. (1984) Science 224,1343-1346 Hanahan, D., and Meselson, M. (1983) Methods Enzymol. 100,333342 Hennecke, H., Springer, M., and Bock, A. (1977) Mol. Gen. Genet. 152,205-210 Hountondji, C., Blanquet, S., and Lederer, F. (1985) Biochemistry 24,1175-1180 Hountondji, C., Dessen, P., and Blanquet, S. (1986a) Biochimie 6 8 , 1071-1078 Hountondji, C., Lederer, F., Dessen, P., and Blanquet, S. (1986b) Biochemistry 25,16-21 Hountondji, C., Schmitter, J.-M., Beauvallet, C., and Blanquet, S. (1987) Biochemistry 26,5433-5439 Huang, S., Elliott, R. C., Liu, P. S., Koduri, R. K., Weickmann, J. L., Lee, J. H., Blair, L. C., Ghost-Dastidar, P., Bradshaw, R. A., Bryan, K. M., Einarson, B., Kendall, R. L., Kolacz, K. H., and Saito, K. (1987) Biochemistry 26,8242-8246 Jordana, X., Chatton, B., Paz-Weisshaar, M., Buhler, J. M., Cramer, F., Ebel, J. P., and Fasiolo, F. (1987) J. Biol. Chem. 2 6 2 , 71897194 Kanehisa, M., Klein, P., Greif, P., and De Lisi, C. (1984) Nucleic Acids Res. 12, 417-428 Khodyreva, S. N., Moor, N. A., Ankilova, V. N., and Lavrik, 0.I. (1985) Biochim. Biophy~.Acta 830,206-212 Koerner, T . J., Myers, A. M., Lee, S., and Tzagoloff, A. (1987) J. Bid. Chem. 262,3690-3696 Laemmli, U. K. (1970) Nature 227,680-685 Maniatis, T., Fritsch, E.F., and Sambrook, J. (1982) Molecular
a and 0 Subunit Genes of Yeast Phenylalanyl-tRNA Synthetase Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SDring Harbor. NY Marczyniki, T., and Jaehning, J. A. (1985) Nucleic Acids Res. 13, 8487-8506 Mechulam, Y.,Fayat, G., and Blanquet, S. (1985) J. Bacterid. 163, 787-791 Mirande, M., Le Corre, D.,Riva, M., and Waller, J. P. (1986) Biochimie 68, 1001-1007 Pailliez, J.-P., and Waller, J.-P. (1984) J. Bwl. Chem. 269, 1549115496 Potier, S., Robbe-Saul, S., and Boulanger, Y. (1980)Biochim. Biophys. Acta 624.130-141 Renaud, M.;Fasiolo, F., Baltzinger, M., Boulanger, Y., and Remy, P.
E.
15415
(1982) Eur. J. Biochem. 123,267-274 Robbe-Saul, S., Fasiolo, F., and Boulanger, Y. (1977) FEBS Lett.84, 57-62 Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. sei. .' .' A. 7 4 95436-5467 Schimmel, P., and Soll, D.(1979)Annu. Reu. Bwchem. 48,601-648 Schimmel, P. (1987) Annu. Reu. Biochem. 66,125-158 2951-2961 Staden, R. (1982) NucleicAcids Res. Tanaka, W. K., Som, K., and Hardesty, B. (1976) Arch. Biochem. ~ i ~ , , h , 172, , ~ , 252-260 Thomas, P. S. (1980) Proc. Natl. Acad. Sci. U. S. A . 77,5201-5205 Van Beveren, C., Straaten, F. V., Curren, T., Muller, R., and Verma, T.M. (1983) Cell -~ 32., 1241-1255 VieirafJ., and Messing, J. (1982) Gene (Amst.) 19, 259-268
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