glutamyl-trna synthetases of bacillus subtilis … · glutamyl-trna synthetases of bacillus...

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THE JOURNAL OF BKXOGKAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 265, No. 30. Issue of October 25, pp. 16246-16255,199O Printed in U.S. A. Glutamyl-tRNA Synthetases of Bacillus subtilis 168T and of Bacillus stearo thermophilus CLONING AND SEQUENCING OF THE gltX GENES AND COMPARISON WITH OTHER AMINOACYL-tRNA SYNTHETASES* (Received for publication, March 14, 1990) Rock Breton$§, David Watsonn, Makoto Yaguchill, and Jacques Lapointe$l[ From the $Dt?partement de Biochimie, Fucultt des Sciences et de Ginie, Universitt Lava& Quibec, GlK 7P4, Canada and the nNationa1 Research Council of Canada, Division of Biological Sciences, 100 Sussex Drive, Ottawa, Ontario KlA OR6, Canada The glutamyl-tRNA synthetase (GluRS) of Bacillus subtilis 168T aminoacylates with glutamate its homol- ogous tRNAG’” and tRNAG’” in viva and Escherichia coli tRNAIG’” in vitro (Lapointe, J., Duplain, L., and Proulx, M. (1986) J. Bacterial. 165,88-93). The gltX gene encoding this enzyme was cloned and sequenced. It encodes a protein of 483 amino acids with a M, of 55,671. Alignment of the amino acid sequences of four bacterial GluRSs (from B. subtilis, Bacillus stearo- thermophilus, E. coli, and Rhizobium meliloti) gives 20% identity and reveals the presence of several short highly conserved motifs in the first two thirds of these proteins. Conserved motifs are found at corresponding positions in several other aminoacyl-tRNA synthe- tases. The only sequence similarity between the GluRSs of these Bacillus species and the E. coli glutam- inyl-tRNA synthetase (GlnRS), which has no counter- part in the E. coli GluRS, is in a segment of 30 amino acids in the last third of these synthetases. In the three- dimenstional structure of the E. coli tRNAGL”.GlnRSe ATP complex, this conserved peptide is near the anti- codon of tRNAG’” (Rould, M. A., Perona, J. J., 5611, D., and Steitz, T. A. (1989) Science 246, 1135-1142), suggesting that this region is involved in the specific interactions between these enzymes and the anticodon regions of their tRNA substrates. The covalent attachment of amino acids to their cognate tRNAs catalyzed by the aminoacyl-tRNA synthetases (aaRSs)’ is one of the crucial steps in the accuracy of protein biosynthesis. Bacterial cells generally contain one aaRS for each amino acid species incoporated into proteins (Grunberg- Manago, 1987); reported exceptions are the presence of two * This work was supported by Grant A-9597 from the National Sciences and Engineering Research Council of Canada and Grant EQ-1700 from the Fondsbour la Formation de Chercheurs et l’Aide 1 la Recherche du Gouvernement du Qu6bec (to J. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adver- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in thispaper has been submitted to the GenBankTMIEMBL Data Bank with accession number(s) 505647. § Postgraduate fellow from FCAR. II To whom correspondence should be addressed: Dept. de Bio- chimie, Facultk des S&ences et de G6nie, Pavillon Vachoni Universitb Laval. Qukbec GlK 7P4. Canada. Tel.: 418-656-3411. ’ The-abbreviations used are: aaRS, aminoacyl-tRNA synthetase; GlnRS, glutaminyl-tRNA synthetase; GluRS, glutamyl-tRNA syn- thetase; TyrRS, tyrosyl-tRNA synthetase; kbp, kilobase pair(s). lysyl-tRNA synthetases in Escherichia coli (Hirshfield et al., 1981), of two threonyl-tRNA synthetases in Bacillus subtilis (Putzer et al., 1990), and the absence of a glutaminyl-tRNA synthetase (GlnRS) in Gram-positive bacteria (Wilcox, 1969a). In one of the latter, B. subtilis, both tRNA”” and tRNAG’” are efficiently aminoacylated with glutamate (Wilcox and Nirenberg, 1968) by the same glutamyl-tRNA synthetase (GluRS) (Lapointe et al., 1986). The mischarged Glu-tRNAG’” is transformed into Gln-tRNA”” by a specific amidotransfer- ase (Wilcox, 1969b). The GluRS and the GlnRS are also linked by the fact that, together with the arginyl-tRNA synthetase, they are the only aaRSs from E. coli which are unable to catalyze the formation of an aminoacyl-adenylate in the absence of their cognate tRNAs (Sal and Schimmel, 1974). Moreover, glutamine and arginine are produced biosynthetically from glutamate (Reitzer and Magasanik, 1987; Glansdorff, 1987). According to the theory of the co-evolution of the genetic code and of the amino acid biosynthetic pathways, glutamine and arginine occupy triplet codons allocated previously to glutamate, one of the few amino acids presumed to have been present in primordial proteins (Wong, 1975; for a review see Wong, 1988). Several groups of aaRSs specific for amino acids linked in their biosynthesis or/and occupying contiguous codons in the genetic code (the GluRS and the GlnRS (Breton et al., 1986), the leucyl-, isoleucyl-, methionyl-, and valyl-tRNA synthetases (Tzagoloff et al., 1988; Heck and Hatfield, 1988) and the aspartyl- and the lysyl-tRNA synthetases (Gampel and Tzagoloff, 1989)) present sequence similarities suggesting a common evolutionary history. In this context, the compar- ison of the primary structures of the GluRS and the GlnRS from E. coli with that of the GluRS of Gram-positive bacteria may reveal structural elements involved in the catalysis of the aminoacylation reaction, in particular those involved in tRNA recognition. In this report, we describe the cloning and the determina- tion of the nucleotide sequences of the B. subtilis and Bacillus stearothermophilus gltX genes, the alignment of the primary structures of the GluRSs from B. subtilis, B. stearother- mophilus, E. coli, and Rhizobium meliloti, and their compari- son with those of the E. coli, Saccharomyces cereuisiae, and human GlnRSs. We have identified a conserved amino acids sequence, shared only by the GluRSs of B. subtilis and of B. stearothermophilus and the GlnRS of E. coli, which may be involved in the interaction between these enzymes and the anticodon region of their tRNA substrates. In the crystal structure of the E. coli glutaminyl-tRNA synthetase com- plexed with tRNAG’” and ATP (Rould et al., 1989), residues 18248 by guest on September 23, 2018 http://www.jbc.org/ Downloaded from

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Page 1: Glutamyl-tRNA Synthetases of Bacillus subtilis … · Glutamyl-tRNA Synthetases of Bacillus subtilis 168T and of ... from E. coli with that of the GluRS of Gram-positive bacteria

THE JOURNAL OF BKXOGKAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 30. Issue of October 25, pp. 16246-16255,199O Printed in U.S. A.

Glutamyl-tRNA Synthetases of Bacillus subtilis 168T and of Bacillus stearo thermophilus CLONING AND SEQUENCING OF THE gltX GENES AND COMPARISON WITH OTHER AMINOACYL-tRNA SYNTHETASES*

(Received for publication, March 14, 1990)

Rock Breton$§, David Watsonn, Makoto Yaguchill, and Jacques Lapointe$l[ From the $Dt?partement de Biochimie, Fucultt des Sciences et de Ginie, Universitt Lava& Quibec, GlK 7P4, Canada and the nNationa1 Research Council of Canada, Division of Biological Sciences, 100 Sussex Drive, Ottawa, Ontario KlA OR6, Canada

The glutamyl-tRNA synthetase (GluRS) of Bacillus subtilis 168T aminoacylates with glutamate its homol- ogous tRNAG’” and tRNAG’” in viva and Escherichia coli tRNAIG’” in vitro (Lapointe, J., Duplain, L., and Proulx, M. (1986) J. Bacterial. 165,88-93). The gltX gene encoding this enzyme was cloned and sequenced. It encodes a protein of 483 amino acids with a M, of 55,671. Alignment of the amino acid sequences of four bacterial GluRSs (from B. subtilis, Bacillus stearo- thermophilus, E. coli, and Rhizobium meliloti) gives 20% identity and reveals the presence of several short highly conserved motifs in the first two thirds of these proteins. Conserved motifs are found at corresponding positions in several other aminoacyl-tRNA synthe- tases. The only sequence similarity between the GluRSs of these Bacillus species and the E. coli glutam- inyl-tRNA synthetase (GlnRS), which has no counter- part in the E. coli GluRS, is in a segment of 30 amino acids in the last third of these synthetases. In the three- dimenstional structure of the E. coli tRNAGL”.GlnRSe ATP complex, this conserved peptide is near the anti- codon of tRNAG’” (Rould, M. A., Perona, J. J., 5611, D., and Steitz, T. A. (1989) Science 246, 1135-1142), suggesting that this region is involved in the specific interactions between these enzymes and the anticodon regions of their tRNA substrates.

The covalent attachment of amino acids to their cognate tRNAs catalyzed by the aminoacyl-tRNA synthetases (aaRSs)’ is one of the crucial steps in the accuracy of protein biosynthesis. Bacterial cells generally contain one aaRS for each amino acid species incoporated into proteins (Grunberg- Manago, 1987); reported exceptions are the presence of two

* This work was supported by Grant A-9597 from the National Sciences and Engineering Research Council of Canada and Grant EQ-1700 from the Fondsbour la Formation de Chercheurs et l’Aide 1 la Recherche du Gouvernement du Qu6bec (to J. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adver- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in thispaper has been submitted to the GenBankTMIEMBL Data Bank with accession number(s) 505647.

§ Postgraduate fellow from FCAR. II To whom correspondence should be addressed: Dept. de Bio-

chimie, Facultk des S&ences et de G6nie, Pavillon Vachoni Universitb Laval. Qukbec GlK 7P4. Canada. Tel.: 418-656-3411.

’ The-abbreviations used are: aaRS, aminoacyl-tRNA synthetase; GlnRS, glutaminyl-tRNA synthetase; GluRS, glutamyl-tRNA syn- thetase; TyrRS, tyrosyl-tRNA synthetase; kbp, kilobase pair(s).

lysyl-tRNA synthetases in Escherichia coli (Hirshfield et al., 1981), of two threonyl-tRNA synthetases in Bacillus subtilis (Putzer et al., 1990), and the absence of a glutaminyl-tRNA synthetase (GlnRS) in Gram-positive bacteria (Wilcox, 1969a). In one of the latter, B. subtilis, both tRNA”” and tRNAG’” are efficiently aminoacylated with glutamate (Wilcox and Nirenberg, 1968) by the same glutamyl-tRNA synthetase (GluRS) (Lapointe et al., 1986). The mischarged Glu-tRNAG’” is transformed into Gln-tRNA”” by a specific amidotransfer- ase (Wilcox, 1969b).

The GluRS and the GlnRS are also linked by the fact that, together with the arginyl-tRNA synthetase, they are the only aaRSs from E. coli which are unable to catalyze the formation of an aminoacyl-adenylate in the absence of their cognate tRNAs (Sal and Schimmel, 1974). Moreover, glutamine and arginine are produced biosynthetically from glutamate (Reitzer and Magasanik, 1987; Glansdorff, 1987). According to the theory of the co-evolution of the genetic code and of the amino acid biosynthetic pathways, glutamine and arginine occupy triplet codons allocated previously to glutamate, one of the few amino acids presumed to have been present in primordial proteins (Wong, 1975; for a review see Wong, 1988). Several groups of aaRSs specific for amino acids linked in their biosynthesis or/and occupying contiguous codons in the genetic code (the GluRS and the GlnRS (Breton et al., 1986), the leucyl-, isoleucyl-, methionyl-, and valyl-tRNA synthetases (Tzagoloff et al., 1988; Heck and Hatfield, 1988) and the aspartyl- and the lysyl-tRNA synthetases (Gampel and Tzagoloff, 1989)) present sequence similarities suggesting a common evolutionary history. In this context, the compar- ison of the primary structures of the GluRS and the GlnRS from E. coli with that of the GluRS of Gram-positive bacteria may reveal structural elements involved in the catalysis of the aminoacylation reaction, in particular those involved in tRNA recognition.

In this report, we describe the cloning and the determina- tion of the nucleotide sequences of the B. subtilis and Bacillus stearothermophilus gltX genes, the alignment of the primary structures of the GluRSs from B. subtilis, B. stearother- mophilus, E. coli, and Rhizobium meliloti, and their compari- son with those of the E. coli, Saccharomyces cereuisiae, and human GlnRSs. We have identified a conserved amino acids sequence, shared only by the GluRSs of B. subtilis and of B. stearothermophilus and the GlnRS of E. coli, which may be involved in the interaction between these enzymes and the anticodon region of their tRNA substrates. In the crystal structure of the E. coli glutaminyl-tRNA synthetase com- plexed with tRNAG’” and ATP (Rould et al., 1989), residues

18248

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gltX Genes for Glutamyl-tRNA Synthetases of Two Bacillus Species

FIG. 3. Nucleotide and deduced amino acid sequences of B. subtilis and B. stearothermophilus gltX. The complete sequences of B. subtilis gltX (Bsu) are given, and differences be- tween them and those of B. stearotherm- ophilus (Bst) sequences are listed on two lines below. Series of three dots indicate gaps. The six internal fragments and NH,- and COOH-terminal segments of the B. subtilis GluRS, whose amino acid sequences have been determined, are ouerlined. (We have previously reported an NH*-terminal sequence of Met-Asn- Glu- . for the B. subtilis GluRS (Proulx et al., 1983). Close examination of the sequence analysis data from which the latter sequence was deduced shows both methionine and glycine to be present in the first cycle and asparagine and glycine in the second cycle. The presence of gly- tine in cycles one and two was, therefore, mistakenly attributed to its presence as a contaminant in either the preparation or the analytical system. In retrospect, the previous data can now be interpreted as the GluRS having two NHz-terminal sequences of 1) NHS-Met-Gly-Asn-Glu-

. and 2) NHp-Gly-Asn-Glu- . . . This led to our mistaken NH,-terminal as- signment of Met-Asn-Glu- . . . )

from this conserved sequence are near the anticodon of tRNA”‘” and may interact with it.

EXPERIMENTAL PROCEDURES’

RESULTS AND DISCUSSION

Partial Sequencing of B. subtilis Glutamyl-tRNA Synthe- tuse-As no mixed-oligonucleotide designed from the NH, terminus of the glutamyl-tRNA synthetase of B. subtilis (Proulx et al., 1983) gave clear hybridization signal with the B. subtilis DNA, we sequenced internal peptides of the en- zyme. Purified GluRS was digested with trypsin and six internal fragments were isolated and sequenced (see Fig. 3).

’ Portions of this paper (including “Experimental Procedures,” part of “Results,” Table I, and Figs. 1 and 2) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

One of them, rich in residues corresponding to a small number of codons, was used to design one 50-mer unique ((Y 5) and two 17-mer mixed (a 5.1 and LY 5.2) oligonucleotides (Table I). In the 50-mer oligonucleotide, the base assignment for the uncertain positions (degeneracy of the code) was done by using the statistical codon usage frequency of B. subtilis (Ogasawara, 1985; Shields and Sharp, 1987).

The NHp-terminal amino acid sequence, as deduced from the nucleotide sequence, is Met-G&Asn-Glu- . . . (Fig. 3). Amino acid sequence analysis of the protein indicates the NH,-terminal sequence to be Gly-Asn-Glu- . . . It appears that GluRS is synthesized with a NH*-terminal methionine residue which is subsequently totally or partially removed. Digestion of the GluRS with carboxypeptidase A released isoleucine, asparagine, and small amounts of lysine and leucine. These are the last four amino acids in the COOH-terminal sequence (-Arg-Leu-Lys-Asn-Ile-COOH) deduced from the nucleotide sequence (shown in Fig. 3). As expected from the known

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18250 gltX Genes for Glutamyl-tRNA Synthetases of Two Bacillus Species

FIG. 4. Amino acid alignments of glutamyl- and glutaminyl-tRNA synthetases. First, the glutamyl-tRNA synthetase sequences from R. neliloti (RmeCluRS), E. coli (EcoGluRS), B. sub- tilis (BsuCluRS), and B. stearothermo- philus (BstCluRS) were aligned by the GAP program (cf. “Experimental Pro- cedure”). Identical or conserved posi- tions were boxed (gray boxes at positions where at least three GluRSs have an identical amino acid and white boxes for positions where at least three GluRSs have similar amino acids (Schwartz and Dayhoff, 1979)). Segments (I, II, and III) where we found, in over 60% of the po- sitions, an identical amino acid in at least three GluRSs are indicated by bi- directional arrows. Short highly con- served motifs (motifs a-g) between the GluRSs are identified by (CIII). Then, each of the glutaminyl-tRNA synthetase sequences (E. coli (EcoGlnRS), human (HumGlnRS), and Saccharomyces cere- uisiae (SceGlnRS)) were individually compared with the GluRSs alignment. Gray boxes were extended to positions where an identity was found for one of the GlnRSs with at least three of the GluRSs. White boxes were extended to positions where a similar amino acid is found for one of the GlnRSs and at least three conserved amino acids of the GluRSs. The short highly conserved mo- tifs between the GlnRSs are identified by (m). The levels of identity between these GluRSs are: Rme-Eco = 34%; Rme- Rsu = 38%; Rme-Bst = 38%; Eco-Bsu = 39%; Eco-Bst = 37%, and Bsu-Bst = 67%. Over the aligned part (G’*-Tz9’), the E. coli GlnRS is about 20% identical to each of the four GluRSs.

specificity of carboxypeptidase A, arginine was not released. Cloning and Sequencing of the gltX Gene of B. subtilis-

Chromosomal DNA was digested with restriction endonucle- ases, separated by electrophoresis, and transferred to a nylon blotting membrane. Hybridization with the radiolabeled 50- mer oligonucleotide (Y 5 at 48 “C gave at least one strong signal and a few weaker signals for each digestion (Fig. 1A). With oligonucleotide (Y 5.1 numerous weak signals appeared for every digestion (results not shown). Hybridization with probe LY 5.2 revealed for every digestion a unique and strong signal at approximately the same position as that of the stronger signal obtained with 01 5 (Fig. 1B).

We isolated DNA fragments of about 1.4 kbp generated by total digestion of chromosomal DNA with HindIII, including a fragment hybridizing with probe 01 5.2, and cloned them into vector PBS. We isolated four hybrid plasmids which had the same physical map (taking into account the orientation of the insert) and chose one of them, pLQB205, for further study. The sequencing strategy for pLQB205 is presented at the top of Fig. 2A. The nucleotide sequence of the entire insert is 1407 base pairs (158 base pairs of which are upstream from the ATG initiation codon) and encodes 418 amino acids of

the GluRS, including its NH, terminus. The remainder of the gltX gene was identified by hybridization of the labeled insert of pLQB205 to a Southern transfer of genomic DNA digested with EcoRV (see Fig. 1C). Since the EcoRV site is near the beginning of the probe relative to the gltX gene, the stronger of the two hybridization signals (about 2.7 kbp, cf. Fig. 1C) was expected to contain the 3’-end of gltX. We also cloned the other hybridizing fragment to make sure that we had the regulatory region upstream gltX. An outline of the sequencing strategy for pLQB204 and pLQB206 is illustrated at the bottom of Fig. 2A. The entire inserts of these two plasmids were sequenced in order to determine the 3’-end sequence of the gltX gene together with the flanking sequences likely to play a role in its regulation (Fig. 3).3 The complete sequence of the gltX gene is presented in Fig. 3. The reading frame is 483 codons long and encodes the six internal peptides isolated from the B. subtili-s GluRS. The four amino acids released by partial digestion of the GluRS with carboxypeptidase A (Ile, Asn, Lys, and Leu) (see above) correspond to the four codons upstream of the end of gltX, indicating that translation of the

j R. Breton and J. Lapointe, unpublished results.

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gltX Genes for Glutamyl-tRNA Synthetases of Two Bacillus Species 18251

FIG. 5. Drawing of the a-carbon backbone of the E. coli glutaminyl-tRNA synthetase in its complex with tRNAG’” (- - -) and ATP (checked) (Rould et al., 1989). Conserved motifs between the various GlnRSs (motifs u-g; see text and Fig. 4) are indicated by heavy lines. The conserved sequence between the E. coli GlnRS and the GluRSs of the two Bacillus species is indicated by an arrow.

GluRS messenger probably stops at the ochre codon shown in Fig. 3.

The gltX gene encodes a polypeptide whose molecular weight (55,671) has a significantly lower value than that (65,500) predicted by the mobility of the GluRS during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Proulx et al., 1983). Such a discrepancy has already been reported for many proteins (Burton et al., 1981; Price et al., 1983; Gitt et al., 1985; and Iacangelo et al., 1986) having a high negative charge density (Gitt et al., 1985), which is the case for the B. subtilis GluRS (Glu and Asp represent 17% of the amino acids).

The codon usage for the highly expressed genes of B. subtilis, such as those for ribosomal proteins, is biased (Shields and Sharp, 1987) although less than that of E. coli. For gltX, codon usage is not typical of the highly expressed genes. The G + C content of the third positions is higher for gltX (42%) and for the weakly expressed genes (44%) than for highly expressed genes (28%) (Shields and Sharp, 1987).

Cloning and Sequencing of the gltX Gene of B. stearother- mophilus-The gltX gene of B. steurothermophilus was cloned using as a probe the insert of pLQB205 encoding the first 418 amino acids of the B. subtilis GluRS. This probe was used to hybridize on Southern blots of B. steurothermophilus DNA digested with various endonucleases. Hybridization at 42 “C in the presence of 25% formamide gave only one or two strong signals per digestion (Fig. 1D). The presence of two signals in the EcoRV digest (about 2.6 and 1.5 kbp) indicated that this

site was in gltX. The 2.6-kbp EcoRV fragment was cloned into PBS, giving plasmid pLQB538. This fragment was mapped, partially sequenced to localize gltX, and used to clone the remainder of the gene by walking on the chromo- some. The double digestion of the genomic DNA with Sac1 and PstI produced a fragment (about 1.8 kbp) that hybridizes with the insert of pLQB538 (Fig. 1E). It was cloned into PBS, giving plasmid pLQB537. The strategy for determining the nucleotide sequence of the inserts of pLQB538 and pLQB537 is presented in Fig. 2B. The gltX gene of B. steurothermophilus encodes a polypeptide of 489 amino acids (Mr 56,183) which is very similar to the B. subtilis GluRS (Fig. 3).

Contrarily to the situation in B. subtilis, the codon usage for B. steurothermophilus genes is very biased. The codon usage for gltX shows a strong bias for a set of codons (habit- ually for those having a G or a C at the third position) and broadly resembles the codon usage found for the few B. steurothermophilus aminoacyl-tRNA synthetase genes stud- ied so far (Winter et al., 1983; Barstow et al., 1986; Borgford et al., 1987).

Primary Structure Alignments of the GluRSs from B. sub- tilis, B. stearothermophilus, E. coli, and R. meliloti-The glu- tamyl-tRNA synthetase is the first aaRS whose primary struc- ture is known for four evolutionary distant procaryotic orga- nisms: B. subtilis and B. stearothermophilus (this paper), E. coli (Breton et al., 1986), and R. meliloti (Laberge et al., 1989). Comparison of these sequences allowed the identification of several highly conserved segments that may be part of the active site or of substrate binding sites.

Fig. 4 shows the best primary sequence alignment of these four GluRSs obtained using the computer program GAP (cf. “Experimental Procedures”). By introducing only a few gaps at scattered places, the four sequences have an identity of 20%, whereas 44% of the positions are identical for at least three GluRSs (gray boxes in Fig. 4). If we take into account the amino acids having similarity in their functional groups (Schwartz and Dayhoff, 1979), 39% of the overall positions are conserved by the four GluRSs and 67% by at least 3 of them (white and gray boxes in Fig. 4). This alignment reveals three long segments (Z, ZZ, and ZZZ) where, in over 60% of the positions, an identical amino acid was present in at least three out of four proteins. These segments are interrupted by two intersegments with no significant similarity (less than 25% of the positions are identical for at least three GluRSs).

Segment I, from I3 to E13’ of the E. coli GluRS, covers the most conserved part among the GluRSs. Indeed, in a region (corresponding to 13-R4’ of the E. coli GluRS) near the NH*- terminal end of each GluRS, 83% (38/46) of the residues are identical for at least three GluRSs and 48% (22/46) for the four enzymes. Segment II goes from RI74 to N307 of the E. coli GluRS and is the longest similar region between the four GluRSs. Finally, segment III is located near the COOH- terminal end and requires a few gaps to achieve the best alignment. Using the E. coli GluRS primary structure as reference, the three segments cover 317 amino acids (out of 471), and the four GluRS sequences have, in these regions, an identity of 30% whereas 62% of the residues are identical in at least 3 of them. This suggests that these parts of the GluRSs are likely to play a role in catalytic activity.

Primary Structure Comparison between the Four GluRSs and the E. coli, Saccharomyces cerevisiae, and Human Glutum- inyl-tRNA Synthetases-To the alignment of the GluRSs, we have added the primary structures of the E. coli (Yamao et al., 1982), S. cerevisiae (Ludmerer and Schimmel, 1987) and human glutaminyl-tRNA synthetases (Thoemmes et al., 1988) in order to find conserved elements indicative of a close

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ses.

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e se

quen

ces

corre

spon

d to

or ar

e pa

rt of

the

highly

co

nserv

ed

motifs

(m

otifs

a, b,

d, e,

f, an

d g)

obse

rved

by

comp

aring

the

pr

imar

y str

uctur

es

of se

vera

l Gl

uRSs

(cf

. Fig

. 4)

. Th

e nu

mber

s in

brack

ets

indica

te

the

numb

er

of am

ino

acids

se

para

ting

motif

a fro

m the

NH

, en

d of

the

synth

etase

or

sepa

ratin

g tw

o mo

tifs.

Dash

ed

boxe

s ind

icate

ho

molog

y be

twee

n mo

tifs

for

amino

acyl-

tRNA

sy

ntheta

ses

spec

ific

for

differ

ent

amino

ac

ids.

Abbr

eviat

ed

name

s for

aa

RSs

are:

ZleR

S,

isoleu

cyl-tR

NA

synth

etase

; Le

t&S,

leu

cyl-tR

NA

synth

etase

; Me

t&S,

me

thion

yl-tR

NA

synth

etase

; Va

lRS,

va

lyl-tR

NA

synth

etase

; Ty

rRS,

tyr

osyl-

tRNA

sy

ntheta

se;

TrpR

S,

trypto

phan

yl-tR

NA

synth

etase

. Ab

brev

iated

na

mes

for

orga

nisms

ar

e: Ec

o, E.

coli

; Rm

e, R.

me

liloti;

Bs

u, B.

su

btilis

; Bs

t, B.

ste

aroth

ermo

philu

s; Bc

a, B.

ca

ldoten

ax;

See,

S.

cer

euisi

ue;

Hum,

hu

man.

Zle

RS(E

co),

Web

ster

et al.

(19

84);

ZleRS

(Sce

), En

glisc

h et

al.

(198

7);

LeuR

S(Ec

o),

(Hae

rtlein

an

d Ma

dern

(1

987)

; Le

uRS(

Sce)

, Tz

agolo

ff et

al.

(198

8);

MetR

S(Ec

o),

Barke

r et

al.

(198

2a);

MetR

S(Sc

e),

Walt

er

et al.

(1

983)

; Va

lRS(

Bst),

Bo

rgfor

d et

al.

(198

7);

ValR

S(Ec

o),

Haer

tlein

et al.

(1

987)

; Va

lRS(

Sce)

, Jo

rdan

a et

al.

(198

7);

Z’yrR

S(Bc

a),

Jone

s et

al.

(198

6);

T’rRS

(Bst)

, W

inter

et

al.

(198

3);

TyrR

S(Ec

o),

Barke

r et

al.

(198

2b);

‘Z’rp

RS(B

st),

Barst

ow

et a2

. (1

986)

; Tr

pRS(

Eco)

, Ha

ll et

al.

(198

2);

Z’rpR

S(Sc

e),

Myers

an

d Tz

agolo

ff (1

985)

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gltX Genes for Glutamyl-tRNA Synthetases of Two Bacillus Species 18253

evolutionary linkage between these two enzymes. Such an alignment between the GluRS and GlnRS of E. coli has already been reported (Breton et al., 1986). Nevertheless, the availability of primary structures for both enzymes from many evolutionary distant organisms permits a better alignment over a longer sequence.

Fig. 4 shows this alignment which goes from the beginning of the GluRSs sequences and covers more than the NH2- terminal half of these proteins (269/471 amino acids for the E. coli GluRS) with only a few insertions or deletions. Con- sidering only positions where at least two GlnRSs have an identical or a similar (with values 2 0.5 in the Dayhoff table (Schwartz and Dayhoff, 1979) resealed by Gribskov and Bur- gess (1986)) amino acid with at least two GluRSs, 44% of all positions in this region are conserved. These conserved posi- tions are especially grouped in a region corresponding to the first 110 amino acids of the E. coli GluRS where the level of conserved residues reaches 63%. Finally in the GlnRSs se- quences, the motif VLSKR) matches that found in the GluRS (KLSKR). When we align these two motifs, a sequence, conserved among the GlnRSs (GWDDPRL), matches with that of GluRSs (GYLPEAL). From this position on, no sig- nificant homology was detected in the COOH-terminal half between the four GluRSs and three GlnRSs, even when allowing many gaps. The alignment covers entirely the two halves of the dinucleotide binding domain and the acceptor binding domain and about half of the helical subdomain in the three-dimensional structure of the E. coli GlnRS (Rould et al., 1989). The strong homology in this region suggests that the tertiary structure of the NHp-half of the GluRS is similar to that reported for the GlnRS.

Considering that B. subtilis GluRS aminoacylates effi- ciently its own tRNAG’” in uivo and one of the two tRNAG1” isoacceptors of E. coli in vitro (Lapointe et al., 1986), any conserved regions between the GluRSs of the two Bacillus species and the E. coli GlnRS which are not conserved with the GluRSs of E. coli and R. meliloti could be involved in the recognition and binding of tRNAG’“. The only region endowed with this property is a sequence of 30 amino acids in B. subtilis (N326-E354) and B. stearothermophilus (N327-D355) GluRSs, similar to the N3g7-D42” region in the COOH-terminal half of the E. coli GlnRS (Fig. 4). Indeed, 45% of the GlnRS residues are identical with those of at least one of these two GluRSs; including the similar amino acids (see above), 72% of this sequence is conserved. Interestingly, in the three-dimensional structure of the GlnRS, this sequence belongs to the distal p- barrel domain, a region found to be at close proximity to the anticodon nucleotides of tRNAG’” (Rould et al., 1989; see heavy line indicated by an arrow in Fig. 5). Since these nucleotides play a role in the recognition of tRNA”‘” by the GlnRS, this homology between the GlnRS of E. coli and the GluRSs of these Gram-positive bacteria indicates that conserved resi- dues in this region of 30 amino acids are probably involved in specific interactions between these enzymes and the antico- don regions of their tRNA substrates. Residues located in loops at the beginning and near the middle of this conserved sequence are in the vicinity of the anticodon of tRNAy’” (Rould et al., 1989 and Fig. 5). These interactions may be significant for the discrimination by the glutaminyl-tRNA synthetase between tRNA”‘” and tRNAG’” since nucleotide 36 is G in tRNA”‘” and C in tRNAG’“, and since at position 34 the three tRNAs recognized in vitro by the B. subtilis GluRS, i.e. B. subtilis tRNAE:“c and tRNAgkG, and E. coli tRNA:kh have a U34, whereas E. coli tRNAEEz and E. coli tRNA& which are not recognized in vitro by this GluRS, have, respec-

tively, a C34 and a modified U34 (Lapointe et al., 1986). Identification of Conserved Sequences among a Group of

Aminoacyl-tRNA Synthetases-Alignment of the four GluRSs reveals the presence of seven short and highly conserved stretches of amino acids (motifs a-g) (Fig. 6), all localized in the first two segments (see Fig. 4). These motifs could repre- sent functionally conserved regions. Two of them (motifs a and f, correspond to regions of homology already reported for other aminoacyl-tRNA synthetases: the signature sequence (Webster et al., 1984) and the consensus KMSKS sequence (Hountondji et al., 1986), respectively. Such highly conserved motifs are also present in other aaRSs whose primary struc- ture is known for several organisms. Comparison of the rela- tive locations and amino acid sequences of these motifs could be indicative of the functions of these motifs and of evolu- tionary relations among these enzymes.

The binding site for tyrosine on the tyrosyl-tRNA synthe- tase (Brick and Blow, 1987; Brick et al., 1988) is formed by residues from several regions of the enzyme. These residues mostly belong to four short sequences highly conserved among TyrRSs from various species. When the homologous signature and KMSKS sequences of the GluRSs are aligned with those of the TyrRSs, the four short sequences occupy sites corre- sponding to motifs a, b, d, and e of the GluRSs (Fig. 6). The first two sequences (corresponding to motifs a and b) are near the NH*-terminal end of all TyrRSs and are separated by 17 amino acids, whereas the other two (motifs d and e), separated by 10 amino acids, are 20 amino acids upstream of a conserved sequence similar to KMSKS. A similar arrangement is found for several other synthetases (Fig. 6). If these synthetases have a three-dimensional structure analogous to that of the TyrRS, these motifs could occupy the same positions and could have the same function, as demonstrated for one residue located in motif b) of the E. coli isoleucyl-tRNA synthetase’ (Fig. 6) and shown to be involved in isoleucine binding (Bur- baum et al., 1989). By analogy with structural studies of the tyrosyl- and isoleucyl-tRNA synthetases, it is possible that the conserved motifs a, b, d, and e could form the binding site for glutamate on GluRS.

The amino acid sequences of these motifs could be indica- tive of an evolutionary linkage between aaRSs specific for distinct amino acid substrates. Indeed, subsets of at least 8 aaRSs, which share the signature and the KMSKS sequences, also share other motifs (Fig. 6). Motif b from isoleucyl-, leu- cyl-, methionyl-, and valyl-tRNA synthetases is very con- served, suggesting that these enzymes form a subclass of synthetases that evolved from the same ancestor (Tzagoloff et al., 1988, Heck and Hatfield, 1988). The homology between glutamyl- and glutaminyl-tRNA synthetases (motifs b, d, and e), is still stronger and is consistent with a close evolutionary linkage between these two enzymes. In the crystal structure of the glutaminyl-tRNA synthetase complexed with tRNA”‘” and ATP (Rould et al., 1989), motifs a, b, d, e, and fare near the ATP or the 3’-end of tRNAG’” (Fig. 5). Motifs c and g are at some distance from these substrates, but their conservation suggests that they could play a role in the catalytic activity of the glutaminyl-tRNA synthetase and probably also in that of the GluRS.

Acknowledgments-We thank S. Laberge and H. Putzer for their help and discussions throughout our multiple attempts at cloning aaRS genes from heterologous organisms, Y. V. Brun and M. Rould for helpful discussions, and C. Lemieux for synthesizing oligonucle- otides.

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18254 gltX Genes for Glutamyl-tRNA Synthetases of Two Bacillus Species

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gltX Genes for Glutamyl-tRNA Synthetases of Two Bacillus Species

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R Breton, D Watson, M Yaguchi and J Lapointewith other aminoacyl-tRNA synthetases.

stearothermophilus. Cloning and sequencing of the gltX genes and comparison Glutamyl-tRNA synthetases of Bacillus subtilis 168T and of Bacillus

1990, 265:18248-18255.J. Biol. Chem. 

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