Eur. J. Biochem. 204,465-472 (1992)
0FEBS 1992
Glutamyl-tRNA synthetase from Thermus thermophilus HB8 Molecular cloning of the gltX gene and crystallization of the overproduced protein Osamu NUREKI I , Kenji SUZUKI Miki HARA-YOKOYAMA', Toshiyuki KOHNO ', Hiroshi MATSUZAWA3, Takahisa OHTA3, Toshiyuki SHIMIZU4, Kosuke MORIKAWA4, Tatsuo MIYAZAWA4 and Shigeyuki YOKOYAMA'
'
Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Japan Department of Physiology, Nihon University School of Dentistry at Matsudo, Japan Depdrlment of AgJiCUltUJal Chemistry, Faculty of Agriculture, University of Tokyo, Japan Protein Engineering Research Institute, Osaka, Japan
(Received October 17, 1991) - EJS 91 1389
The gene for the Glu-tRNA synthetase from an extreme thermophile, Thermus thermophilus HB8, was isolated using a synthetic oligonucleotide probe coding for the N-terminal amino acid sequence of Glu-tRNA synthetase. Nucleotide-sequence analysis revealed an open reading frame coding for a protein composed of 468 amino acid residues ( M , 53901). Codon usage in the T. thermophilus GlutRNA synthetase gene was in fact similar to the characteristic usages in the genes for proteins from bacteria of genus Thermus:the G C content in the third position of the codons was as high as 94%. In contrast, the amino acid sequence of T . thermophilus Glu-tRNA synthetase showed high similarity with bacterial Glu-tRNA synthetases (35-45% identity); the sequences of the binding sites for ATP and for the 3' terminus of tRNAG'" are highly conserved. The Glu-tRNA synthetase gene was efficiently expressed in Escherichia coli under the control of the tac promoter. The recombinant T. thermophilus Glu-tRNA synthetase was extremely thermostable and was purified to homogeneity by heat treatment and three-step column chromatography. Single crystals of T. thermophilus Glu-tRNA synthetase were obtained from poly(ethy1ene glycol) 6000 solution by a vapor-diffusion technique. The crystals diffract X-rays beyond 0.35 nm. The crystal belongs to the orthorhombic space group P212121,with unit-cell parameters of a = 8.64 nm, h = 8.86 nm and c = 8.49 nm.
+
Aminoacyl-tRNA synthetases strictly recognize both the cognate tRNA species and amino acids to guarantee correct translation of codons into amino acid residues. Glu-tRNA synthetase, as well as Gln-tRNA synthetase and Arg-tRNA synthetase of Escherichia coli require the cognate tRNA to bind with the cognate amino acids and to catalyze ATP/PPi exchange reactions [l]. Furthermore, we have shown that GlutRNA synthetase from Thermus thermophilus, an extremely thermophilic Gram-negative bacterium [2], binds both Lglutamate and D-glutamate in the absence of tRNAG'", whereas in the presence of tRNAG'",the Glu-tRNA synthetase specifically binds L-glutamate [3]. Previous studies using the methods of CD 13, 41 and fluorescence [3] have revealed that the conformations of Glu-tRNA synthetase and tRNAG'" change upon cognate complex formation (mutual adaptation), Correspondence to S. Yokoyama, Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, Japan 113 Enzymes. Glutamyl-tRNA synthetase (EC 6.1.1.17); methionyllRNA synthetase (EC 6.1.1 .lo); glutaminyl-tRNA synthetase (EC 6.1.1.18); arginyl-tRNA synthetase (EC 6.1.1.19); tyrosyl-tRNA synthetase (EC 6.1.1. l ) ; phenylalanyl-tRNA synthetase (EC 6.1.1.20); threonyl-tRNA synthetase (EC 6.1.1.3).
which may explain the functions of tRNAG'" as an allosteric modulator 13, 51. However, it remains to be elucidated how the mutual adaptation induces the activation of the catalytic site of the synthetase. A s for E. coli Gln-tRNA synthetase, cocrystals of the synthetase with tRNAG1"and ATP have been analyzed at 0.28 nm resolution, and it has been shown that the conformations of the acceptor stem and the anticodon loop of tRNAG'" change drastically upon complex formation [6]. This Gln-tRNA synthetase shows high amino acid sequence similarity with E. coli Glu-tRNA synthetase in the N-terminal half 171. In fact, Bacillus subtilis and some eukaryotic Glu-tRNA synthetases charge glutamic acid to homologous tRNAG'" species in vivo [8,9]. In contrast, the C-terminal halves of GlutRNA synthetases show low sequence similarity with the GlntRNA synthetase [9]. The only exception is that B. subtilis Glu-tRNA synthetase, which misacylates E. coli tRNAG1", has been shown to have insertion of a sequence homologous with E. coli Gln-tRNA synthetase for the putative binding site of the anticodon of the tRNAG1"[lo]. This suggests that the tRNA-recognition mechanism of Glu-tRNA synthetase is different from that of Gln-tRNA synthetase.
466 To elucidate the mechanism of tRNA recognition by GlutRNA synthetase and of the mutual adaptation upon complex formation, the tertiary structure of Glu-tRNA synthetase and of the complex with the cognate tRNA and other substrates should be analyzed using physicochemical techniques such as X-ray crystallography and NMR spectroscopy. In extending such structural analyses further, thermostable Glu-tRNA synthetase from thermophilic bacteria is more useful than the enzyme from mesophilic bacteria, as shown by previous studies on Bacillus stearothermophilus Tyr-tRNA synthetase [ l l ] and T. thermophilus Phe-tRNA, Ser-tRNA and ThrtRNA synthetases [12- 141. In the present study, we have cloned and overexpressed a gene (gltX) coding for Glu-tRNA synthetase from T. thermophilus. This Glu-tRNA synthetase, overproduced in E. coli cells, is extremely thermostable and expected to be suitable for structural analyses. In fact, we have succeeded in crystallizing the thermophile Glu-tRNA synthetase.
MATERIALS AND METHODS Preparation of Glu-tRNA synthetase from T.thevmophilus T. thermophilus strain HB8 (ATCC 27634) [2] was kindly provided by Dr T. Oshima (Tokyo Institute of Technology). Glu-tRNA synthetase was purified from T. thermophilus almost to homogeneity by chromatography on columns of DEAE-cellulose, DEAE-Sephacel, phosphocellulose and hydroxyapatite as described previously [15]. This Glu-tRNA synthetase preparation was applied to a TSK-Gel G3000SW HPLC column (Tosoh) equilibrated with 200 mM potassium phosphate, pH 7.0 and elution was performed with the same buffer at a flow rate of 0.5 ml/min 1151. Enzyme activity T. thermophilus Glu-tRNA synthetase was assayed at 65 "C by the aminoacylation reaction of E. coli tRNAG'" with L-[U''C]glutamic acid (Amersham) as described previously [I 51. N-terminal amino acid sequence analysis The N-terminal amino acid sequence of the purified T. thermophilus Glu-tRNA synthetase was determined with an Applied Biosystems gas protein sequencer model 470A.
alkaline phosphatase, the DNA-ligation kit, the nick-translation kit and the kilo-sequence kit were from Takara Shuzo. E. coli strain MVI 184 { A (srl-recA)306 : : TnIO, A ( I N proAB), ara-, thi-, rpsL-, q580dlacZAM15 [F':proAB, I d q , lacZAM15, traD3q) was used as the host for screening of the T. thermophilus gltX gene by colony hybridization and as the host for expression of the T. thermophilus gltX gene. The detailed methods used for molecular cloning were based on those of Maniatis et al. [16]. Chromosomal DNA from T. thermophilus HB8 was prepared by the method of Marmur [17]. The Southern-blot filters were used for hybridization with a 5'-labeled 26-nucleotide probe and a 0.95-kbp DNA fragment which was labeled by nick-translation and were washed with 0.1 x 0.15 M NaCl and 0.15 M sodium citrate (NaCliCit.) at 50°C and at 6 5 ° C respectively. DNA sequencing DNA fragments, subcloned into pUC11S and pUC139, were sequenced by the dideoxy-chain termination method [I 81 with KO7 helper phage and a 7-deaza-Sequenase kit (US Biochemical Corp.). For computer analysis of the nucleotide and amino acid sequences, a primary-sequence-analysis software package Genetyx (Software Development Co. Ltd.) was used. Construction of recombinant plasmids for expression of the T. thermophilusgltX gene in E. coli A 1.9-kbp A h 1 - Hind111 DNA fragment containing T. thermophilus gltX gene (Fig. 1) was subcloned into the SmaI/ Hind111 sites of plasmids pUC118, pUCl19 and pEXP7 (pUC118 and pUC119 having a lac promoter and a highcopy-number pEXP7 with a tac promoter) [19], resulting in pUCIlS/gltX1.9, pUC119/gltX1.9, and pEXP7/gltX1.9, respectively (Fig. 1). Oligonucleotide-directed mutagenesis of the glrX gene Plasmid pEXP7/gltX1.9 was digested with BamHI and the resultant 0.5-kbp BamHI fragment was subcloned into the BamHI site of MI 3mp19. Oligonucleotide-directed mutagenesis was performed on the MI3 DNA template with a Muta-Gene in vitro mutagenesis kit (Bio Rad Laboratories) by the method of Kunkel [20]. Thus, the mutated BamHI fragment was ligated with a 1.4-kbp fragment of BamHIdigested plasmid pEXP7/gltX1.9.
Oligonucleotide synthesis and 32Plabeling A mixed oligodeoxyribonucleotideprobe was synthesized by the phosphoramidite method on a DNA synthesizer (model 380A, Applied Biosystems) and purified by chromatography with an oligonucleotide purification cartridge (Applied Biosystems) according to the instruction of the supplier. The oligonucleotide (50 pmol) was 5'-labeled at 37°C in a volume of 25 p1 with 5 U T4 polynucleotide kinase (Takara Shuzo, Kyoto, Japan) and 25 nmol [y-32P]ATP (5000 Ci/mmol; Amersham) and further purified by electrophoresis on denaturing 12% polyacrylamide gels (20 x 20 x 0.1 cm). The labeled oligonucleotide probe was used for Southern hybridization. Molecular cloning of the T. thermophilusgItX gene Restriction enzymes were purchased from Toyobo Biochemicals (Osaka, Japan) and from Takara Shuzo. Bacterial
Overproduction of T. thermophilus Glu-tRNA synthetase in E. coli
E. coli MV1184 cells carrying the recombinant plasmid were cultured at 37°C for 2 h in a medium (pH 7.0 with NaOH) containing 20 g/1 tryptone, 8 g/1 NaCI, 1 pg/ml thiamine and 50 pg/ml ampicillin. Isopropyl-j-thiogalactoside was added to a concentration of 0.5 mM and incubation was continued for 6 h. The cells were harvested by centrifugation. Cell extract The harvested cells were suspended in 50 mM Tris/HCl, pH 7.9 (1.5 ml/g wet cells) containing 10 mM magnesium acetate, 10 mM 2-mercaptoethanol and 0.5 mM phenylmethanesulfonyl fluoride. After ultrasonic disruption of the cells, crude cell extract was obtained by centrifugation (30 min at 10000 xg) and incubated at 70°C for 20 min. Thus, most of
KpnI
--
---
I -
c* -~ t --
t-
---
-
-+
IAluI, Hindm
.-.--
467
A -A
--
-
c
~
_ _+
-
-+
Fig. 2. Restriction map of the T. fhermophilusgltxgene and sequencing strategy. Most of the sequence was determined by sequencing deletion mutants of the gltX gene with a kilo-sequence kit. The open arrow indicates the T. therrnophilus gltX gene. K, KpnI; B, BumHI; 13, HindIII; S, S a d ; (A), the Nu1 site described in Materials and Methods.
UHd glt x 1.8
Fig.1. Construction of the plasmids used for expression of the T. thermophilusgltX gene. The 5.2-kbp KpnI fragment containing the T . therrnophilus gltX gene was ligated into the Kpnl site of plasmid pUCl18 to produce pUCl18/gltX5.2. By partial digestion of the 5.2-kbp KpnI fragment, a 1.9-kbp AluI -Hind111 fragment was obtained and was subcloned into pEXP7, pUC118 and pUC119 to produce pEXP7/gltX1.9, pUCl lS/gltXl.9 and pUCll9/gltXl.9. respectively. pEXP7/gltX1.8 was obtained from pEXP7/gltX1.9 through deletion OF 68-nucleotides upstream of the gltX gene by oligonucleotide-directed mutagenesis. Bm, BurnHI site; Hd, Hind111 site; Al, AluI site; Sm, SrnaI site; Amp', the p-lactamase gene; solid arrow, the gltX gene; Plac, E. coli, lac promoter; Ptac, E. coli tuc promoter.
E. coli proteins were denatured and removed from the cell extract by centrifugation at I0000 x g for 30 min. Finally, the supernatant mainly containing T. thermophilus Glu-tRNA synthetase was obtained. Purification of T. thermophilus Glu-tRNA synthetase overproduced in E. coli The heat-treated extract of 180 g E. coli MV1184 cells carrying pEXP7/gltXI .8 (Fig. 1) was dialyzed twice against 3 1 50 mM Tris/HCl, pH 7.9, containing 20 mM NH,Cl, and the dialysate was applied to a DEAE-Sephacel ion-exchange column (7 cm x 20 cm). The Glu-tRNA synthetase was eluted at a flow rate of 1.6 ml/min with 2 1 of a linear gradient of NH,Cl of 10-100 mM. The eluted fraction, mainly containing T. thermophilus Glu-tRNA synthetase, was further applied to a Mono Q HR 30/10 FPLC column (Pharmacia) equilibrated with 20 mM Tris/HCl, pH 7.4, containing 10 mM magnesium acetate and 3 0 mM 2-mercaptoethanol. Elution was performed at a flow rate of 4 ml/min with a linear gradient of KCl(0 - 200 mM) in the same buffer. Collected fractions were dialyzed against 50 mM potassium phosphate, pH 7.0, and applied to a phenyl-Superose H R 20/10 FPLC column (Pharmacia). Elution was performed at a flow rate of I ml/ min with a linear gradient of ammonium sulfate (3 -0 M). The protein concentration was determined according to the modified Lowry method [22], using bovine serum albumin as a standard.
Crystallization procedure
The purified Glu-tRNA synthetase was dialyzed at 4°C against 10 mM Tris/HCl, pH 7.0, containing 30 mM 2-mercaptoethanol. Crystallization was carried out by the vapor-diffusion method of the hanging-drop mode [23]. Glassdepression plates were siliconized with Silicofilm (Nacdlai Tesque Co. Ltd.) and mounted in Linbro multi-well tissueculture plates (Flow Laboratories). Crystallization drops (10-20 pl) were equilibrated at 12°C against 1-ml reservoirs. Various crystallization conditions were tested as described in Results and Discussion, changing the salt concentration, pH, precipitants, protein concentration, divalent cations and reducing agents. Crystal handling Crystals were harvested in a stabilizing solution containing 60 mM Mes/KOH, pH 6.5, 20% poly(ethy1ene glycol) 6000, 2.4 mM MgC12, 12 mM 2-mercaptoethanol, 1.2 mM NaN, and 1.2% (by vol.) 2-methylpentane-2,4-diol. For SDS/ PAGE, crystals were washed several times with fresh mother liquor and dissolved in a small volume of distilled water. For X-ray diffraction, crystals were mounted in Lindenmann glass capillaries (Hilgenberg Glass, Malsfeld, Germany). X-ray diffraction Diffraction patterns were obtained with a precession camera (Enraf-Nonius) and an automated oscillation camera system (DIP-100, MAC-Science) [24, 251 using an imaging plate, which were operated on a rotating-anode X-ray generator.
RESULTS AND DISCUSSION Molecular cloning and sequencing of T. thermophilusgltX gene By Edman degradation, the N-terminal amino acid sequence up to the 40th residue of Glu-tRNA synthetase purified from T . thermophilusHB8 was determined to be M'VVTRIAPSPTGDPHVGTAYIALFNYAWARRNGGRFIVRI. A mixed 26-oligonucleotide probe 5'-d(GACCCECACG 'r",GGEACgGCgTACAT)-3' coding for the amino acid sequence DI3PHVGTAYI was synthesized so that the third letter of each codon was G and/or C in accordance with the
468 CCCCGGAGAGGGCCCGTAGGCCATCCGGTCCCGCAGGTGGT~GATGGAAAGCTCCTCCAAAAGGGCCTTGGCCTTCTCCAGGCGCTCCTTTTTGGAAAGGGGCTGGAACTCCAAGACGGC - 3 6 1 Smal -24 1
GAGGAGGTTCTCCAAGGCGGTCATCCGCCGGAAGGCGCTGGGCTCCTGGGGGAGGTAGCCAGGGCCCCCGGGCCGGTTTGTACATGGGGAGGGCGGTGATCTCCTGGCCCTTAAGGTAGA BamHI
BanHI
-121
TCCGCCCCCCCGTGGGCCGGATGAACCCCACCACCATGTAGAAGGTGGTGGGCTTCCCCGGATGGTTGGGGATCCGAAAGAGGGATCCCACGATCTCGCCGCCCTAAGGGGCGAGGTCCA Smal
SD
CGCCCCGGACCACCTCCTTCGGGCGTAGGCCTTGCGCAAACCCTGGGCCCGAAGCTCCCCGTCCATTGCCCCTTAGGCTACGGGGCAACCTTTAGCCCCGGGTGAGATAATGGGC~T
-1
ATGGTGGTGACCCGCATCGCGCCAAGCCCCACGl;GCGACCCCCACGTGG[~CACGGCCTACATCGCCCTCTTCAACTACGCCTGGGCCCGGAGGAACGGGGGGCGCTTCATCGTGCGCATT 120 M V V T R I A P S P T G D P H V G T A Y I A L F N Y A W A R R N G G R F I V R I 40 Smoi BadI GAGGACACGGACCGGGCGCGCTACGTCCCCGGGGCCGAGGAAAGGATCCTCGCCGCCTTAAAGTGGCTTGGCCTCTCCTACGACGAGGGCCCCGATGTGGCGGCCCCCACGGGCCCCTAC E D T D R A H Y V P G A E E R I L A A L K W L G L S Y D E G P D V A A P T G P Y
240 80
Sac1
CGCCAGTCGGAACGGCTTCCCCTCTACCAAAAGTACGCCGAGGAGCTCCTCAAGCGGGGGTGGGCCTACCGGGCCTTTGAGACCCCGGAGGAGCTAGAGCAAATCCGCAAGGAAAAGGGG R Q S E R L P L Y Q K Y A E E L L K R G W A Y R A F E T P E E L E Q I R K E K G
360 120
Smn I
GGCTACGACGGGAGGGCCCGGAACATCCCCCCTGAGGAGGCCGAGGAGCGGGCGAGGCGGGGCGAGCCCCACGTGATCCGCCTCAAGGTGCCCCGCCCCGGGACCACGGAGGTCAAGGAC
C
Y
D
C
R
A
R
N
I
P
P
E
E
A
E
E
R
A
R
R
G
E
P
H
V
I
R
L
K
V
P
R
P
G
T
T
E
V
K
D
480 160
SacI
GAGCTCAGGGGGGTGGTGGTCTACGACAACCAGGAGATCCCCGACGTGGTCCTCCTCAAGTCCGACGGCTACCCCACCTACCACCTGGCCAACGTGGTGGACGACCACCTCATGGGGGTC E L R G V V V Y D N Q E I P D V V L L R S D G Y P T Y H L A N V V D D H L M G V
600 200
ACGCACGTGATCCGGGCGGAGGAGTGGCTCGTCTCCACCCCCATCCACGTCCTCCTCTACCGGGCCTTCGGCTGGGAGGCGCCCCGGTTCTACCACATGCCCCTCCTGCGCAACCCCGAC 720 T D V I R A E E H L V S T P I H V L L Y R A F G H E A P R F Y H M P L L R N P D 240
AAGACCAAGATCTCCAAGCGCAAAAGCCACACCTCCCTGGACTGGTACAAGGCGGAGGGGTTTCTGCCCGAGGCCCTGAGGAACTACCTCTGCCTCATGGGGTTCTCCATGCCCGACGGG K T K I S K R K S H T S L D W Y K A E G F L P E A L R N Y L C L M G F S M P D G
840 280
CCCCACATCTTCACCCTCGAGGAGTTCATCCACGCCTTCACCTGGGAGAGGGTTTCCCTGGGGGGGCCCGTCTTTGACCTGGAAAAGCTCCGCTGGATGAACGGGAAG~ACATCCGGGAG R E I F T L E E F I Q A F T W E R V S L G G P V F D L E K L R H M N G K Y I R E
960
320
Sac1
GTGCTCTCCCTGGAGGAGGTGGCGGAGAGGGTCAAGCCCTTCCTCCGGGAGGCGGGGCTTTCCTGGGAAAGCGAGGCCTACCTGAGGCGGGCGGTGGAGCTCATGCGCCCCCGGTTTGAC V L S L E E V A E R V K P F L R E A G L S W E S E A Y L R R A V E L M R P R F D
I080 360
Sac1 ACCCTGAAGGAGTTCCCGGAGAAGGCCCGCTACCTCTTCA~CGAGGACTACCCCGTGTCGGAGAAGGCCCAGAGGAAGCTGGAAGAGGGGCTTCCCCTCCTCAAGGAGCTCTACCCCCGC
1200
T
R
400
CTAAGGGCCCAGGAGGAGTGGACCGAGGCCCTCGACGCCCTCCTCCGGGGCTTCGCCGCGGAGAAGGGGGTCAAGCTCGGCCAGGTGGCCCAGCCCCTTCGGGCCGCCCTCACGGGG
G
1320 440
AGCCTGGAGACCCCGGGGCTTTTTGAGATCCTGGCCCTCCTCGGGAAGGAGCGGGCGCTCAGGCGCTTGGAGCGGGCCCTCGCCTAGGGGGTATACTGGACGCCATGAGACGGCTTCTCG S L E T P G L F E I L A L L G K E R A L R R L E R A L A *
1440 468
TCGTCCTTCTCGCCCTTTTGGGCCTCGGCCTCGGCCCAAGGCGCCATCGGGATGCGCTTCGGCTACGGGGAAGGCCTCGGCCTCACCTTCGGGGCCGGGATGGAGAACCGCCTACGGCAG
1560
AACCTCTCGGGCCGCCTCGCCGCCGACCTCGGGCCCGAGGGCCCCGGCCTGGCCCTGGAGGCCCTCCTCCTCTTCAAGCCCGACCTGGGGCAGTACGAGGGCGCCCTCAAAGGCTCCTCC
1680
CCTACGTGGCCGG
1693
L
L
R
R
A
E
F
Q
E
P
E
E
W
K
T
A
E
R
A
Y
A
L
L
F
E
T
A
E
L
D
L
Y
R
P
G
V
F
S
A
E
A
K
E
A
K
Q
G
R
V
K
K
L
L
E
G
E
Q
G
V
L
A
P
Q
L
P
L
L
K
R
E
A
L
A
Y
L
P
T
Sma I
Fig. 3. Nucleotide sequenceof the T.thermophilusgltX gene and the deduced amino acid sequence of Glu-tRNA synthetase. The underlined amino acid scquence was determined by E d m a n degradation and amino acid sequencing. The putative Shine-Dalgarno sequence is indicated.
preferential codon usage in genes from bacteria of the genus Ti~ernius[19, 26- 291. This 26-nucleotide probe was labeled with 32P, and used for Southern hybridization to T. thermophilus chromosomal DNA digested with several restriction enzymes, resulting in marked hybridization to a Sac1 fragment of about 0.95 kbp (data not shown). This 0.95-kbp Sac1 fragment was cloned into the SacI site of plasmid pUCl19 by colony hybridization. The insert was sequenced and found to include the 5'-terminal300 nucleotides of the T. thcrrnophilus g l M gene (described below). In order to clone the full-length DNA of the T. thermophilus gltX gene, the 0.95-kbp insert was labeled by nick-translation and used for Southern hybridization, yielding a single band of a 5.2-kbp KpriI digest. The 5.2-kbp KpnI fragment was cloned into plasmid pUCl18 by colony hybridization. The complete DNA sequence of a 2.4-kbp region of the KpnI fragment was determined on both strands by a sequencing strategy shown in Fig. 2, and the T. thermophilus gltX gene was found to be included in this sequenced region. The nucleotide sequence of the T. therniophilusgltXgene with its flanking regions is shown in Fig. 3.
Amino acid sequence of T. thermophilus Glu-tRNA synthetase
A nucleotide sequence coding for the amino acid sequence of the N-terminal 40 residues of T. thermophilus Glu-tRNA synthetase was found as underlined in Fig. 3. Thus, the open reading frame of the gltX gene was identified to be composed of 1407 bp, coding for 468 amino acid residues as shown in Fig. 3. The relative molecular mass was calculated to be 53 901, in agreement with that estimated previously by SDSi PAGE ( M I 50000) [15]. The amino acid composition of the Glu-tRNA synthetase, estimated from the amino acid sequence, is similar to that obtained for the hydrolysate of purified T. thermophilus Glu-tRNA synthetase [15]. Codon usage in the T. thermophilus gltX gene
+
The G C content at the third position of codons in the T. thermophilus gltX gene was found to be as high as 94%, which is similar to that (94%) of T. thermophilus Met-tRNA synthetuse gene [21] and those (85-96%) in other genes from bacteria of the genus Thermus [19,26- 291. Thus, codon usage
469
116 120 109 119 119
(7. t h . ) (R. me.) ( E . ca.) \B. su.) (B. st.)
349 ( R . re.\
468 ( T. 484 (R. 471 ( E . 483 ( B . 489 ( B .
th.) me.) co.) su.) st.)
Pig.4. Comparison of the amino acid sequences of the Glu-tRNA synthetases from T. therrnophifus, R. rneliloti, E. cofi, B. subrilis and B. siearotherrnophilus. Amino acid residua conserved in morc than three Glu-tRNA synthetases are enclosed by boxes. Identical residues to those of T. thermophilus Glu-tRNA synthetases are also encloscd. Signature sequence and similar sequence to KMSK are underlined. The abbreviations are as follows: T . th., T. thermophilus; R. me., R.meliloti; E. co., E. coli; B. su., B. subtilis; B. st., B. stearothrnnopltilus.
in the T. thermophilus &X' gene is quite different from that in the E. coligltX gene [7]. In particular, codons CGG (arginine), AAG (lysine); GAG (glutamate) are frequently used in the T. thermophilus gltX gene (Fig. 3), while these codons are rarely used in E. coli genes [30, 311. This frequent use of minor codons in the T. therrnophilus gltX gene would be a problcm in overexpression of the gltX gene in E. coli cells.
Amino-acid-sequence homology of Glu-tRNA synthetases from T. thermophilus, Rhizobium rneliloti, E. coli, B. subtilis and B. stearothermophilus Amino acid sequences of T. therrnophilus (this work), E. coli [7], R. rneliloti [32], B. suhtilis and B. steurothermophilus
[lo] Glu-tRNA synthetases are well aligned on the basis of homology (Fig. 4), indicating a structural similarity between these Glu-tRNA synthetases. Comparison of the entire amino acid sequences of T. thermophilus Glu-tRNA synthetases with those of E. coli, R. meliloti, B. subtilis and B. stewothermophilus (Fig. 4) reveals amino acid identities of 37%, 45%, 35% and 41 %, respectively and similarities in conservative replacements of 12%, 13%, 8% and 11%, respectively. Thus, T. thermophilus Glu-tRNA synthetase is the most homologous of all species tested to R . rneliloti Glu-tRNA synthetase, possibly because the R. melilotigltXgene [32] has as high a G C content as that of the T. thermophilus gltX gene. In the five Glu-tRNA synthetases, the N-terminal halves show greater similarity than the C-terminal halves (Fig. 4). As
+
470 for T. thermophilus Met-tRNA synthetase also, the N-terminal domain which is involved in the catalysis of aminoacylation shows greater similarity with other Met-tRNA synthetases than the second domain which is involved in tRNA'" recognition. Furthermore, the N-terminal halves of the Glu-tRNA synthetases show amino acid sequence similarity with the E. coli Gln-tRNA synthetase N-terminal half [33], which, by crystallographic analyses, has been shown to interact with ATP and the 3'-terminus of tRNA"'" [6]. Thus, class-I aminoacyl-tRNA synthetases [34], including Met-tRNA synthetase, Gln-tRNA synthetase and Glu-tRNA synthetase are suggested to have at least two functional domains, that is, the N-terminal domain for aminoacylation catalysis and the C-terminal domain for tRNA recognition. Putative ATP-binding site of T. thermophilus Glu-tRNA synthetase
T. thermophilus Glu-tRNA synthetase has a sequence HVG in positions 15-17, which is highly conserved in all the four Glu-tRNA synthetases (Fig. 4). Around the HVG sequence, high similarities are found among the five GlutRNA synthetases. These regions constitute the signature sequences found in several aminoacyl-tRNA synthetases which have consensus HIGH sequences essential for ATP binding [35]. It is interesting to note that the amino acid residue next to the HVG sequence is not conserved among the Glu-tRNA synthetases, unlike other aminoacyl-tRNA synthetases. Putative amino-acid-binding site
We have previously shown that the fluorescence intensity of T. thermophilus Glu-tRNA synthetase is remarkably reduced when the synthetase is bound to L-glutamate [3], suggesting the presence of a tryptophan residue near the amino-acid-binding site. In the N-terminal halves of all the sequenced Glu-tRNA synthetases which were suggested to be involved in aminoacylation catalysis, there is only one tryptophan residue (Trp62 of the T. thermophilus enzyme) conserved among all the five Glu-tRNA synthetases (Fig. 4). Furthermore, a short amino acid segment around this tryptophan is also conserved in Gln-tRNA synthetase from E. coli [33]. Thus, Trp62 of T. thermophilus Glu-tRNA synthetase may be involved in the interaction with L-glutamate. Putative binding site for tRNAG1"
T. thermophilus Glu-tRNA synthetase aminoacylates tRNA"'" from E. coli (Knl0.60 pM) as well as tRNA"" from T. thermophilus ( K , 0.65 pM) in vitro [15]. Thus, T. thermophilus and E. coli Glu-tRNA synthetases have similar tertiary structures for tRNA recognition. Aminoacyl-tRNA synthetases belonging to class I [34] have a highly conserved amino acid sequence whose consensus is KMSKS. This sequence is probably involved in the binding of the 3'-terminus of the cognate tRNA species [36, 371. T. thermophilus GlutRNA synthetase also has a similar sequence KISKR (amino acids 243 - 247), which is well conserved in all five Glu-tRNA synthetases (Fig. 4). In the case of Met-tRNA synthetases from E. coli and T. thermophilus, KMSKS motifs are located near the boundaries of the N-terminal catalytic domain and the second tRNArecognition domain [21]. Thus, like in Met-tRNA synthetases, the sequence KLSKR in Glu-tRNA synthetase is supposed to lie on the boundary of the two functional domains. In the
Fig. 5. SDS/PAGE of T.thermophilus Glu-tRNA synthetase produced in E. coli-carrying recombinant plasmids. Lane 1, relative molecular mass standards (66000, bovine serum albumin; 45000, egg albumin; 36000, glyceraldehyde-3-phosphatedehydrogenase; 29000, carbonic anhydrase; 24000, trypsinogen; 20000, trypsin inhibitor; 14000, z-lactalbumin); lane 2, heat-treated extract of E. coli cells carrying pUC118/gltX1.9; lane 3, pUC119/gltX1.9; lane 4, pEXPIJlgltX1.9; lane 5, pEXP7/gltX1.8. T. thermophilus Glu-tRNA synthetase is indicated by an arrow.
putative C-terminal domain, there are hghly conserved amino acid residues among all the sequenced Glu-tRNA synthetases. In the T. thermophilus Glu-tRNA synthetase sequence, the amino acid regions 259-266 and 305-314 are possibly involved in specific interaction with tRNAG'". In this context, we have previously found that the fluorescence intensity of T . thermophilus Glu-tRNA synthetase at 350 nm is reduced to 75% when the Glu-tRNA synthetase binds to T. thermophilus or E. coli tRNAG'" (31, suggesting the presence of a tryptophan residue near the tRNA-binding site. In fact, in the putative Cterminal domain, only one tryptophan residue (Trp312 in the T. thermophilus enzyme) is conserved among all the five GlutRNA synthetases (Fig. 4). Substitutions of Trp312 have been shown to reduce the aminoacylation activity of the Glu-tRNA synthetase (unpublished results). Furthermore, from a I3CNMR analysis on l3C-labe1ed tRNAG1",we have found that an aromatic amino acid residue of T. thermophilus Glu-tRNA synthetase is involved in interaction with the first nucleotide of the anticodon of tRNA"'" (unpublished result). Thus, we propose that the conserved tryptophan residue (Trp312 of T. thermophilus Glu-tRNA synthetase) is involved in recognition of the anticodon region of tRNAG'". Expression of the T. thermophilusgltX gene in E. coli
In order to elucidate the structure/function relationship of the thermostable Glu-tRNA synthetase by physicochemical techniques, a system for large-scale preparation of the enzyme should be established. Therefore, to overproduce the GlutRNA synthetase in E. coli, the gltX gene was inserted into various expression vectors (Fig. 1). E. coli cells transformed with pUC118/gltX1.9 and pEXP7/gltX3.9 produced thermostable T. thermophilus GlutRNA synthetase (Fig. 5, lanes 2 and 4), while E. coli cells transformed with pUC119/gltX1.9 did not (Fig. 5, lane 3). This indicates that, in the former cases, the T. thermophilus gltX gene was expressed from the lac and tac promoter in the presence of lac inducer. In SDS/PAGE, the extract of the pUCl18/gltX1.9 transformant showed one more band, the molecular mass of which was slightly higher than that of T. thermophilus Glu-tRNA synthetase. This thermostable product appears to be a fusion protein of the N-terminal region of j?-lactamase with T. therrnophilus Glu-tRNA synthetase. Furthermore, we deleted 68 nucleotides upstream of the initiation codon of the gltX gene on pEXPTJ/gltXI.9 so that the
Fig.6. The 12% S D S / P A W monitoring the purification and crystalllxation of 7. thrvmophilus C.lu-tRNA synthetase produced in E. colicarrjing pEXP7/gltXI.S. Lanl: I. ccll f r e lysatc; lane 2. lysatc after heat treatment at 70 C for 20 min: lane 3. T . rhcrmnphilus GlutRNA synthetasc purified by thrcc-step column chromatography (sce Materials and Methods); lane 4, a single-washcd crystal. The band of T.r/iermophilua (ilu-t R K A synthetase is indicatcd by :in arrow.
klg.7. Microphotograph O f crystals O f T. thermophiius GIU-tRYA synthetase. The scale bar reprcscnts 0.1 mm.
glrX mRNA was translated with the effective E. coli ShincDalgarno scqucncc (pEXP7/gltX1.8; Fig. 1). Thus. T.ther)nophilus Glu-tKNA synthetase was cfficiently overproduced in E . coli (Fig. 5. lane 5).
Purification of T. thermophilus Glu-tRNA synthetase overproduced in E. coli cells A heat-trcated extract of E. coli cclls, carrying pEXP7I gltX1.8. showcd one major band corresponding to T . rhcrrnophilus Glu-tRNA synthetasc on SDS/PAGE (Fig. 6, lane 2). Thus. heat-treatment is shown to be a useful step for purification of T. thermophilus Glu-t RNA synthctasc from I:. coli to hornogencity, as in the case of T. f h e r ~ n o p h i l uMet~ tRNA synthetase 121). We then purified 7'. rherntophilus Glut R N A synthetase from the E. coli ccll extract by chromatography on columns of DEAE-Sephacel. MonoQ and phenylSuperose, according to Materials and Mcthods (Fig. 6, lane 3). Thus, we obtained 50 mg pure T. ~hrrniophilusGlu-tKNA synthetase from 180 g E. roli cells. On SL)S,'PAGE, the mobility of the purified 7.. thrrmophilus Glu-tRNA synthetase, overproduced in E. coli cclls, was equal to that of the Glut RNA synthetase purified from 7'. thermophilus cclls (data not shown). Furthermore. the purified T . tlicvniophilus Glu-tKNA synthetase overproduced in E. c o l i w x B S active as the cn7yme from T. ~hzrinoppldu.~ cells in aruinoacylation of tRNAG'" species from E. c d i and T. fhermophihts (data not shown). Thus, the thermostable Glu-tRNA synthetase overproduced in E. co/i will be uscful for physicochcmical studies by NMK speclroscopy and X-ray crystallography.
Crystallimtion of T. thermophilus Glu-tRNA synthetase Crystalli7ation was carried out at 1 2 ' C under various conditions: the compo4tion of the precipitant 125 - 53% saturated ammonium sullate and 11 - 22% poly(ethy1ene glycol) 6000 and poly(cthylenc glycol) 30001. thc composition and pH of thc buffcr (SO mM 'L'ris,'acetate for pH 7.0 - 7.8.40 mM potassium phosphate for pH 6.5 - 7.5, SO mM Mes for pH 6.0 - 7.0, and SO mM Bistris, 50 m M Bistrisjpropane and 50 mM Pipes l o r pH 6.5) and the concentration of T. rhermophilrrs Glu-t RNA synthetase (2.6 - 10 mg at thc starting point) wcrc varied. In all trials, 2 mM MgClz and 10 mM 2-mercaptoethanol were added. Crystallization with ammonium sulfate and poly(ethylene glycol) 3000 uas unsuc-
Fig. 8. Precesion photographs of (Okl) reciprocal lattice zone. The precession anglc was 15 ' and exposure time was 24 h.
cessful. Ncedlc-like crystals. which had a strong tendency for forming a cluster. were grown from 15 - 18% poly(eth).lt.ne glycol) 6000 ;it pH 6.0-6.6. At highcr concentrations of poly(ethy1ene glyco1)WOO ( 1 9 - - 22%) or at higher pH (6.8 7.8), thinner crystals or an amorphous precipitate appeared. To improve the crystal characteristics. 2-met hylpentane-2.4diol was added to a final concentration of 1 - 6%. according to the observation of Yaremchuk et al. 1131. The butfer used (Mes, Bistris. Bistris/propanc and Pipes) slightly affected the crystal morphology. Best single crystals with dimensions 01' 0 . 1 5 x 0 . 1 5 x l . O m m (Fig. 7) appeared within 2 - 3 weeks at 12°C in a solution containing 5 mg/ml protein. 15. - 1 7 O h poly(cttiylene glyc01)6000, 50 mM Mes. pH 6.2 -6.5. 2 m M MpCIZ. 1 0 m M 2-mercaptoethanol, 1 m,M NaN3 and 1'70 2niethylpentane-2,4-diol. A crystal was washed and subjected t o SDSjPAGE. One major band wits observed with :t mobility corresponding to the intact T. !hcrrnophilus Glu-tRNA synthetase (Fig. 6, lane 4).
Preliminary crystallographic data Crystal data were obtained from precession photographs showing reflections to about 0.25 nni resolution (Fig. 8). The Okl and hol p h e s indicate mmm symmetry and an extinction law of / r = 2 n . k = 2 n. I = 2 n. I t was thus concluded that the
472 crystals belong to the orthorhombic space group P212121 with unit-cell parameters of a = 8.64 nm, b = 8.86 nm and c = 8.49 nm. The unit-cell volume is 6.50 x 10' nm3 and assumed to contain one molecule in an asymmetric unit. The value [38i is therefore to be 3010 nm3/Da>indicating that the crystals have a rather high solvent content. From measurements with a precession camera and DIPloo, the crysta1s were found to diffract to at least O.35-nm resolution. The crystals are stable against X-ray radiation and suitable for X-ray structural analysis. To obtain even better crystals of T. thermophilus Glu-tRNA synthetase and also cocrystals of Glu-tRNA synthetase and tRNAG'", further trials are now in progress.
v,
The authors are grateful to Prof. Akinori Suzuki of the Department of Agricultural Chemistry, University of Tokyo for amino acid sequencing, to Dr Susumu Nishimura of the National Cancer Center Research Institute for the synthesis of the oligodeoxyribonucleotide probe and to Dr Hiroyuki Adachi of the Department of Agricultural Chemistry. University of Tokyo for his helpful discussion. This work was supportcd in part by Grant-in-Aid for Specially Promoted Research (60060004) and Grants-in-Aid for Scientific Research on Priority Areas (01656002 and 02238102) from the Ministry of Education, Science and culture of Japan, by a Bioscience Grant for International Joint Research Project from the New Energy and Jndustrial Technology Devclopment Organization, and by a Research Grant from International Human Frontier Science Program Organization.
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3. Hara-Yokoyama. M., Yokoyama, S. & Miyazawa, T. (1986) Biochemistry 25, 7031 - 7036. 4. Willick, G. E. & Kay, C. M. (1976) Biochemistry I5,4347-4352. 5. Kern, D. & Lapointe. J. (1979) Biochemistry 18, 5819-5826. 6. Rould. M. A., Perona, J . J., SOU, D. & Steitz, T. A. (1989) Science 246. 1135-1142. 7. Breton, R., Sanfxon, H., Papayannopoulos, I., Biemann, K. & Lapointe, J. (1986) J . Biol. Chem. 261, 10630-10617. 8. Proulx, M., Duplain, L., Lacostc, L., Yaguchi, M. & Lapointe, J . (1983) J . B i d . Chem. 258, 753-759. 9. Schon, A., Kannangara, C. G., Cough, S. & SOU, D. (1988) Nature 331, 1 87 - 190. 10. Breton, R.. Watson, D., Yaguchi, M. & Lapointe, J. (1990) J . Biol. Chem. 265, 18248 - 18255. 11. Brick, P., Bhat, T. N. & Blow, D. M. (1988) 1. Mol. Biol. 208, 83 -98. 12. Ankilova, V. N., Reshetnikova, L. S., Chernaya, M. M. & Lavrik, 0. I. (1988) FEBS Lett. 227,Y-13. 13. Yaremchuk. A. D., Tukalo, M. A. & Egorova, S. P. (1990) J . Mol. Biol. 213,631 -632.
14. Garber, M. B., Yaremchuk, A. D., Tukalo, M. A., Egorova, S. P., Fomenkova, N. P. & Nikonov, S. V. (1990) J . Mol. Biol. 214,819-820. 15. Hara-Yokoyama, M., Yokoyama, S. & Miyazawa, T. (1984) J . Biochem. (Tokyo) 96, 1599- 1607. 16. Maniatis, T,, Fritsch, E, F. & Sambreook, J , (1982) Molecular cloning, pp. 326 - 328,382 - 389, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 16a. Maniatis, T., Fritsch, E. F. & Sambreook, J. (1992) Molecular cloning, pp. 382-389, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 17. Marmur, J. (1961) J . Mol. B i d . 3,208-218. 18. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl Acad. Sci. USA 74, 5463 - 5467. 19. Ono, M., Matsuzawa, H. & Ohta, T. (1990) J . Biochon. (Tokyo) 107, 21 -26. 20. Kunkel, T. A. (1985) Proc. Natl Acad. Sci.USA 82,488-492. 21. Nureki, O., Muramatsu, T., Suzuki, K., Kohda, D., Matsuzawa, H., Ohta, T., Miyazawa, T. & Yokoyama, S. (1991) J . B i d . Chem. 266,3268 - 3277. 22. Bensadoun, A. & Weinstein, D. (1976) Anal. Biochem. 70, 241 250. 23. Wlodawer, A. & Hodgson, K. (1975) Proc. Nati Acad. Sci. USA 72,398 - 399. 24. Tanaka, I., Yao, M., Suzuki, K., Matsumoto, T., Kozasa, M. & Katayama, C. (1990) J . Appl. Crystal. 23, 334-339. 25. Fujii, I., Morimoto, Y . ,Higuchi, Y. & Yasuoka, N. (1991) Acta Crystal. B47, 137-144. 26. Kagawa, Y., Nojima, H., Nukiwa, N., Ishizuka, M., Nakajima. T., Yasuhara, T., Tanaka, T. & Oshima, T. (1984) J . Biol. Chem. 259,2956 - 2960. 27. Nishiyama, M., Matsubara, N., Yamamoto, K., Iijima, S., Uozumi, T. & Beppu, T. (1986) J. B i d . Chem. 261, 1417814183. 28. Kunai, K., Machida, M., Matsuzawa, H. & Ohta, T. (1986) Eur. J. Biochem. 160,433-440. 29. Kushiro, A,, Shimizu, M. & Tomita, K. (1987) Eur. J . Biochem. 170,93-98. 30. Ikemura, T. (1981) J . Mol. Biol. 151,389-409. 31. Aota, S., Gojobori, T., Ishibashi, F., Maruyama, T. & Ikemura, T. (1988) Nucleic Acids Res. 16, 315-402. 32. Laberge, S., Gagnon, Y., Bordeleau, L. M. & Lapointe, 3. (1989) J. Bacteriol. 171, 3926 - 3932. 33. Yamao, F., Inokuchi, H., Cheung, A., Ozcki, H. &So11, D. (1982) J. Biol. Chem. 257, 11639- 11 643. 34. Eriani, G., Delarue, M., Poch, O., Gangloff, J. & Moras, D. (1990) Nature 347, 203 - 206. 35. Webster, T. A., Lathrop, R. H. &Smith, T. F. (1 987) Biochemistry 26,6950-6957. 36. Hartlein, M. & Madern, D. (1987) Nucleic Acids Res. 15,1019910210. 37. Hountondji, C., Schmitter, J. M., Beauvallet, C . & Blanquet, S. (1987) Biochemistry 26, 5433 - 5439. 38. Mattews, B. W. (1968) J. Mol. Biol. 33,491 -491.
0FEBS 1992
Glutamyl-tRNA synthetase from Thermus thermophilus HB8 Molecular cloning of the gltX gene and crystallization of the overproduced protein Osamu NUREKI I , Kenji SUZUKI Miki HARA-YOKOYAMA', Toshiyuki KOHNO ', Hiroshi MATSUZAWA3, Takahisa OHTA3, Toshiyuki SHIMIZU4, Kosuke MORIKAWA4, Tatsuo MIYAZAWA4 and Shigeyuki YOKOYAMA'
'
Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Japan Department of Physiology, Nihon University School of Dentistry at Matsudo, Japan Depdrlment of AgJiCUltUJal Chemistry, Faculty of Agriculture, University of Tokyo, Japan Protein Engineering Research Institute, Osaka, Japan
(Received October 17, 1991) - EJS 91 1389
The gene for the Glu-tRNA synthetase from an extreme thermophile, Thermus thermophilus HB8, was isolated using a synthetic oligonucleotide probe coding for the N-terminal amino acid sequence of Glu-tRNA synthetase. Nucleotide-sequence analysis revealed an open reading frame coding for a protein composed of 468 amino acid residues ( M , 53901). Codon usage in the T. thermophilus GlutRNA synthetase gene was in fact similar to the characteristic usages in the genes for proteins from bacteria of genus Thermus:the G C content in the third position of the codons was as high as 94%. In contrast, the amino acid sequence of T . thermophilus Glu-tRNA synthetase showed high similarity with bacterial Glu-tRNA synthetases (35-45% identity); the sequences of the binding sites for ATP and for the 3' terminus of tRNAG'" are highly conserved. The Glu-tRNA synthetase gene was efficiently expressed in Escherichia coli under the control of the tac promoter. The recombinant T. thermophilus Glu-tRNA synthetase was extremely thermostable and was purified to homogeneity by heat treatment and three-step column chromatography. Single crystals of T. thermophilus Glu-tRNA synthetase were obtained from poly(ethy1ene glycol) 6000 solution by a vapor-diffusion technique. The crystals diffract X-rays beyond 0.35 nm. The crystal belongs to the orthorhombic space group P212121,with unit-cell parameters of a = 8.64 nm, h = 8.86 nm and c = 8.49 nm.
+
Aminoacyl-tRNA synthetases strictly recognize both the cognate tRNA species and amino acids to guarantee correct translation of codons into amino acid residues. Glu-tRNA synthetase, as well as Gln-tRNA synthetase and Arg-tRNA synthetase of Escherichia coli require the cognate tRNA to bind with the cognate amino acids and to catalyze ATP/PPi exchange reactions [l]. Furthermore, we have shown that GlutRNA synthetase from Thermus thermophilus, an extremely thermophilic Gram-negative bacterium [2], binds both Lglutamate and D-glutamate in the absence of tRNAG'", whereas in the presence of tRNAG'",the Glu-tRNA synthetase specifically binds L-glutamate [3]. Previous studies using the methods of CD 13, 41 and fluorescence [3] have revealed that the conformations of Glu-tRNA synthetase and tRNAG'" change upon cognate complex formation (mutual adaptation), Correspondence to S. Yokoyama, Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, Japan 113 Enzymes. Glutamyl-tRNA synthetase (EC 6.1.1.17); methionyllRNA synthetase (EC 6.1.1 .lo); glutaminyl-tRNA synthetase (EC 6.1.1.18); arginyl-tRNA synthetase (EC 6.1.1.19); tyrosyl-tRNA synthetase (EC 6.1.1. l ) ; phenylalanyl-tRNA synthetase (EC 6.1.1.20); threonyl-tRNA synthetase (EC 6.1.1.3).
which may explain the functions of tRNAG'" as an allosteric modulator 13, 51. However, it remains to be elucidated how the mutual adaptation induces the activation of the catalytic site of the synthetase. A s for E. coli Gln-tRNA synthetase, cocrystals of the synthetase with tRNAG1"and ATP have been analyzed at 0.28 nm resolution, and it has been shown that the conformations of the acceptor stem and the anticodon loop of tRNAG'" change drastically upon complex formation [6]. This Gln-tRNA synthetase shows high amino acid sequence similarity with E. coli Glu-tRNA synthetase in the N-terminal half 171. In fact, Bacillus subtilis and some eukaryotic Glu-tRNA synthetases charge glutamic acid to homologous tRNAG'" species in vivo [8,9]. In contrast, the C-terminal halves of GlutRNA synthetases show low sequence similarity with the GlntRNA synthetase [9]. The only exception is that B. subtilis Glu-tRNA synthetase, which misacylates E. coli tRNAG1", has been shown to have insertion of a sequence homologous with E. coli Gln-tRNA synthetase for the putative binding site of the anticodon of the tRNAG1"[lo]. This suggests that the tRNA-recognition mechanism of Glu-tRNA synthetase is different from that of Gln-tRNA synthetase.
466 To elucidate the mechanism of tRNA recognition by GlutRNA synthetase and of the mutual adaptation upon complex formation, the tertiary structure of Glu-tRNA synthetase and of the complex with the cognate tRNA and other substrates should be analyzed using physicochemical techniques such as X-ray crystallography and NMR spectroscopy. In extending such structural analyses further, thermostable Glu-tRNA synthetase from thermophilic bacteria is more useful than the enzyme from mesophilic bacteria, as shown by previous studies on Bacillus stearothermophilus Tyr-tRNA synthetase [ l l ] and T. thermophilus Phe-tRNA, Ser-tRNA and ThrtRNA synthetases [12- 141. In the present study, we have cloned and overexpressed a gene (gltX) coding for Glu-tRNA synthetase from T. thermophilus. This Glu-tRNA synthetase, overproduced in E. coli cells, is extremely thermostable and expected to be suitable for structural analyses. In fact, we have succeeded in crystallizing the thermophile Glu-tRNA synthetase.
MATERIALS AND METHODS Preparation of Glu-tRNA synthetase from T.thevmophilus T. thermophilus strain HB8 (ATCC 27634) [2] was kindly provided by Dr T. Oshima (Tokyo Institute of Technology). Glu-tRNA synthetase was purified from T. thermophilus almost to homogeneity by chromatography on columns of DEAE-cellulose, DEAE-Sephacel, phosphocellulose and hydroxyapatite as described previously [15]. This Glu-tRNA synthetase preparation was applied to a TSK-Gel G3000SW HPLC column (Tosoh) equilibrated with 200 mM potassium phosphate, pH 7.0 and elution was performed with the same buffer at a flow rate of 0.5 ml/min 1151. Enzyme activity T. thermophilus Glu-tRNA synthetase was assayed at 65 "C by the aminoacylation reaction of E. coli tRNAG'" with L-[U''C]glutamic acid (Amersham) as described previously [I 51. N-terminal amino acid sequence analysis The N-terminal amino acid sequence of the purified T. thermophilus Glu-tRNA synthetase was determined with an Applied Biosystems gas protein sequencer model 470A.
alkaline phosphatase, the DNA-ligation kit, the nick-translation kit and the kilo-sequence kit were from Takara Shuzo. E. coli strain MVI 184 { A (srl-recA)306 : : TnIO, A ( I N proAB), ara-, thi-, rpsL-, q580dlacZAM15 [F':proAB, I d q , lacZAM15, traD3q) was used as the host for screening of the T. thermophilus gltX gene by colony hybridization and as the host for expression of the T. thermophilus gltX gene. The detailed methods used for molecular cloning were based on those of Maniatis et al. [16]. Chromosomal DNA from T. thermophilus HB8 was prepared by the method of Marmur [17]. The Southern-blot filters were used for hybridization with a 5'-labeled 26-nucleotide probe and a 0.95-kbp DNA fragment which was labeled by nick-translation and were washed with 0.1 x 0.15 M NaCl and 0.15 M sodium citrate (NaCliCit.) at 50°C and at 6 5 ° C respectively. DNA sequencing DNA fragments, subcloned into pUC11S and pUC139, were sequenced by the dideoxy-chain termination method [I 81 with KO7 helper phage and a 7-deaza-Sequenase kit (US Biochemical Corp.). For computer analysis of the nucleotide and amino acid sequences, a primary-sequence-analysis software package Genetyx (Software Development Co. Ltd.) was used. Construction of recombinant plasmids for expression of the T. thermophilusgltX gene in E. coli A 1.9-kbp A h 1 - Hind111 DNA fragment containing T. thermophilus gltX gene (Fig. 1) was subcloned into the SmaI/ Hind111 sites of plasmids pUC118, pUCl19 and pEXP7 (pUC118 and pUC119 having a lac promoter and a highcopy-number pEXP7 with a tac promoter) [19], resulting in pUCIlS/gltX1.9, pUC119/gltX1.9, and pEXP7/gltX1.9, respectively (Fig. 1). Oligonucleotide-directed mutagenesis of the glrX gene Plasmid pEXP7/gltX1.9 was digested with BamHI and the resultant 0.5-kbp BamHI fragment was subcloned into the BamHI site of MI 3mp19. Oligonucleotide-directed mutagenesis was performed on the MI3 DNA template with a Muta-Gene in vitro mutagenesis kit (Bio Rad Laboratories) by the method of Kunkel [20]. Thus, the mutated BamHI fragment was ligated with a 1.4-kbp fragment of BamHIdigested plasmid pEXP7/gltX1.9.
Oligonucleotide synthesis and 32Plabeling A mixed oligodeoxyribonucleotideprobe was synthesized by the phosphoramidite method on a DNA synthesizer (model 380A, Applied Biosystems) and purified by chromatography with an oligonucleotide purification cartridge (Applied Biosystems) according to the instruction of the supplier. The oligonucleotide (50 pmol) was 5'-labeled at 37°C in a volume of 25 p1 with 5 U T4 polynucleotide kinase (Takara Shuzo, Kyoto, Japan) and 25 nmol [y-32P]ATP (5000 Ci/mmol; Amersham) and further purified by electrophoresis on denaturing 12% polyacrylamide gels (20 x 20 x 0.1 cm). The labeled oligonucleotide probe was used for Southern hybridization. Molecular cloning of the T. thermophilusgItX gene Restriction enzymes were purchased from Toyobo Biochemicals (Osaka, Japan) and from Takara Shuzo. Bacterial
Overproduction of T. thermophilus Glu-tRNA synthetase in E. coli
E. coli MV1184 cells carrying the recombinant plasmid were cultured at 37°C for 2 h in a medium (pH 7.0 with NaOH) containing 20 g/1 tryptone, 8 g/1 NaCI, 1 pg/ml thiamine and 50 pg/ml ampicillin. Isopropyl-j-thiogalactoside was added to a concentration of 0.5 mM and incubation was continued for 6 h. The cells were harvested by centrifugation. Cell extract The harvested cells were suspended in 50 mM Tris/HCl, pH 7.9 (1.5 ml/g wet cells) containing 10 mM magnesium acetate, 10 mM 2-mercaptoethanol and 0.5 mM phenylmethanesulfonyl fluoride. After ultrasonic disruption of the cells, crude cell extract was obtained by centrifugation (30 min at 10000 xg) and incubated at 70°C for 20 min. Thus, most of
KpnI
--
---
I -
c* -~ t --
t-
---
-
-+
IAluI, Hindm
.-.--
467
A -A
--
-
c
~
_ _+
-
-+
Fig. 2. Restriction map of the T. fhermophilusgltxgene and sequencing strategy. Most of the sequence was determined by sequencing deletion mutants of the gltX gene with a kilo-sequence kit. The open arrow indicates the T. therrnophilus gltX gene. K, KpnI; B, BumHI; 13, HindIII; S, S a d ; (A), the Nu1 site described in Materials and Methods.
UHd glt x 1.8
Fig.1. Construction of the plasmids used for expression of the T. thermophilusgltX gene. The 5.2-kbp KpnI fragment containing the T . therrnophilus gltX gene was ligated into the Kpnl site of plasmid pUCl18 to produce pUCl18/gltX5.2. By partial digestion of the 5.2-kbp KpnI fragment, a 1.9-kbp AluI -Hind111 fragment was obtained and was subcloned into pEXP7, pUC118 and pUC119 to produce pEXP7/gltX1.9, pUCl lS/gltXl.9 and pUCll9/gltXl.9. respectively. pEXP7/gltX1.8 was obtained from pEXP7/gltX1.9 through deletion OF 68-nucleotides upstream of the gltX gene by oligonucleotide-directed mutagenesis. Bm, BurnHI site; Hd, Hind111 site; Al, AluI site; Sm, SrnaI site; Amp', the p-lactamase gene; solid arrow, the gltX gene; Plac, E. coli, lac promoter; Ptac, E. coli tuc promoter.
E. coli proteins were denatured and removed from the cell extract by centrifugation at I0000 x g for 30 min. Finally, the supernatant mainly containing T. thermophilus Glu-tRNA synthetase was obtained. Purification of T. thermophilus Glu-tRNA synthetase overproduced in E. coli The heat-treated extract of 180 g E. coli MV1184 cells carrying pEXP7/gltXI .8 (Fig. 1) was dialyzed twice against 3 1 50 mM Tris/HCl, pH 7.9, containing 20 mM NH,Cl, and the dialysate was applied to a DEAE-Sephacel ion-exchange column (7 cm x 20 cm). The Glu-tRNA synthetase was eluted at a flow rate of 1.6 ml/min with 2 1 of a linear gradient of NH,Cl of 10-100 mM. The eluted fraction, mainly containing T. thermophilus Glu-tRNA synthetase, was further applied to a Mono Q HR 30/10 FPLC column (Pharmacia) equilibrated with 20 mM Tris/HCl, pH 7.4, containing 10 mM magnesium acetate and 3 0 mM 2-mercaptoethanol. Elution was performed at a flow rate of 4 ml/min with a linear gradient of KCl(0 - 200 mM) in the same buffer. Collected fractions were dialyzed against 50 mM potassium phosphate, pH 7.0, and applied to a phenyl-Superose H R 20/10 FPLC column (Pharmacia). Elution was performed at a flow rate of I ml/ min with a linear gradient of ammonium sulfate (3 -0 M). The protein concentration was determined according to the modified Lowry method [22], using bovine serum albumin as a standard.
Crystallization procedure
The purified Glu-tRNA synthetase was dialyzed at 4°C against 10 mM Tris/HCl, pH 7.0, containing 30 mM 2-mercaptoethanol. Crystallization was carried out by the vapor-diffusion method of the hanging-drop mode [23]. Glassdepression plates were siliconized with Silicofilm (Nacdlai Tesque Co. Ltd.) and mounted in Linbro multi-well tissueculture plates (Flow Laboratories). Crystallization drops (10-20 pl) were equilibrated at 12°C against 1-ml reservoirs. Various crystallization conditions were tested as described in Results and Discussion, changing the salt concentration, pH, precipitants, protein concentration, divalent cations and reducing agents. Crystal handling Crystals were harvested in a stabilizing solution containing 60 mM Mes/KOH, pH 6.5, 20% poly(ethy1ene glycol) 6000, 2.4 mM MgC12, 12 mM 2-mercaptoethanol, 1.2 mM NaN, and 1.2% (by vol.) 2-methylpentane-2,4-diol. For SDS/ PAGE, crystals were washed several times with fresh mother liquor and dissolved in a small volume of distilled water. For X-ray diffraction, crystals were mounted in Lindenmann glass capillaries (Hilgenberg Glass, Malsfeld, Germany). X-ray diffraction Diffraction patterns were obtained with a precession camera (Enraf-Nonius) and an automated oscillation camera system (DIP-100, MAC-Science) [24, 251 using an imaging plate, which were operated on a rotating-anode X-ray generator.
RESULTS AND DISCUSSION Molecular cloning and sequencing of T. thermophilusgltX gene By Edman degradation, the N-terminal amino acid sequence up to the 40th residue of Glu-tRNA synthetase purified from T . thermophilusHB8 was determined to be M'VVTRIAPSPTGDPHVGTAYIALFNYAWARRNGGRFIVRI. A mixed 26-oligonucleotide probe 5'-d(GACCCECACG 'r",GGEACgGCgTACAT)-3' coding for the amino acid sequence DI3PHVGTAYI was synthesized so that the third letter of each codon was G and/or C in accordance with the
468 CCCCGGAGAGGGCCCGTAGGCCATCCGGTCCCGCAGGTGGT~GATGGAAAGCTCCTCCAAAAGGGCCTTGGCCTTCTCCAGGCGCTCCTTTTTGGAAAGGGGCTGGAACTCCAAGACGGC - 3 6 1 Smal -24 1
GAGGAGGTTCTCCAAGGCGGTCATCCGCCGGAAGGCGCTGGGCTCCTGGGGGAGGTAGCCAGGGCCCCCGGGCCGGTTTGTACATGGGGAGGGCGGTGATCTCCTGGCCCTTAAGGTAGA BamHI
BanHI
-121
TCCGCCCCCCCGTGGGCCGGATGAACCCCACCACCATGTAGAAGGTGGTGGGCTTCCCCGGATGGTTGGGGATCCGAAAGAGGGATCCCACGATCTCGCCGCCCTAAGGGGCGAGGTCCA Smal
SD
CGCCCCGGACCACCTCCTTCGGGCGTAGGCCTTGCGCAAACCCTGGGCCCGAAGCTCCCCGTCCATTGCCCCTTAGGCTACGGGGCAACCTTTAGCCCCGGGTGAGATAATGGGC~T
-1
ATGGTGGTGACCCGCATCGCGCCAAGCCCCACGl;GCGACCCCCACGTGG[~CACGGCCTACATCGCCCTCTTCAACTACGCCTGGGCCCGGAGGAACGGGGGGCGCTTCATCGTGCGCATT 120 M V V T R I A P S P T G D P H V G T A Y I A L F N Y A W A R R N G G R F I V R I 40 Smoi BadI GAGGACACGGACCGGGCGCGCTACGTCCCCGGGGCCGAGGAAAGGATCCTCGCCGCCTTAAAGTGGCTTGGCCTCTCCTACGACGAGGGCCCCGATGTGGCGGCCCCCACGGGCCCCTAC E D T D R A H Y V P G A E E R I L A A L K W L G L S Y D E G P D V A A P T G P Y
240 80
Sac1
CGCCAGTCGGAACGGCTTCCCCTCTACCAAAAGTACGCCGAGGAGCTCCTCAAGCGGGGGTGGGCCTACCGGGCCTTTGAGACCCCGGAGGAGCTAGAGCAAATCCGCAAGGAAAAGGGG R Q S E R L P L Y Q K Y A E E L L K R G W A Y R A F E T P E E L E Q I R K E K G
360 120
Smn I
GGCTACGACGGGAGGGCCCGGAACATCCCCCCTGAGGAGGCCGAGGAGCGGGCGAGGCGGGGCGAGCCCCACGTGATCCGCCTCAAGGTGCCCCGCCCCGGGACCACGGAGGTCAAGGAC
C
Y
D
C
R
A
R
N
I
P
P
E
E
A
E
E
R
A
R
R
G
E
P
H
V
I
R
L
K
V
P
R
P
G
T
T
E
V
K
D
480 160
SacI
GAGCTCAGGGGGGTGGTGGTCTACGACAACCAGGAGATCCCCGACGTGGTCCTCCTCAAGTCCGACGGCTACCCCACCTACCACCTGGCCAACGTGGTGGACGACCACCTCATGGGGGTC E L R G V V V Y D N Q E I P D V V L L R S D G Y P T Y H L A N V V D D H L M G V
600 200
ACGCACGTGATCCGGGCGGAGGAGTGGCTCGTCTCCACCCCCATCCACGTCCTCCTCTACCGGGCCTTCGGCTGGGAGGCGCCCCGGTTCTACCACATGCCCCTCCTGCGCAACCCCGAC 720 T D V I R A E E H L V S T P I H V L L Y R A F G H E A P R F Y H M P L L R N P D 240
AAGACCAAGATCTCCAAGCGCAAAAGCCACACCTCCCTGGACTGGTACAAGGCGGAGGGGTTTCTGCCCGAGGCCCTGAGGAACTACCTCTGCCTCATGGGGTTCTCCATGCCCGACGGG K T K I S K R K S H T S L D W Y K A E G F L P E A L R N Y L C L M G F S M P D G
840 280
CCCCACATCTTCACCCTCGAGGAGTTCATCCACGCCTTCACCTGGGAGAGGGTTTCCCTGGGGGGGCCCGTCTTTGACCTGGAAAAGCTCCGCTGGATGAACGGGAAG~ACATCCGGGAG R E I F T L E E F I Q A F T W E R V S L G G P V F D L E K L R H M N G K Y I R E
960
320
Sac1
GTGCTCTCCCTGGAGGAGGTGGCGGAGAGGGTCAAGCCCTTCCTCCGGGAGGCGGGGCTTTCCTGGGAAAGCGAGGCCTACCTGAGGCGGGCGGTGGAGCTCATGCGCCCCCGGTTTGAC V L S L E E V A E R V K P F L R E A G L S W E S E A Y L R R A V E L M R P R F D
I080 360
Sac1 ACCCTGAAGGAGTTCCCGGAGAAGGCCCGCTACCTCTTCA~CGAGGACTACCCCGTGTCGGAGAAGGCCCAGAGGAAGCTGGAAGAGGGGCTTCCCCTCCTCAAGGAGCTCTACCCCCGC
1200
T
R
400
CTAAGGGCCCAGGAGGAGTGGACCGAGGCCCTCGACGCCCTCCTCCGGGGCTTCGCCGCGGAGAAGGGGGTCAAGCTCGGCCAGGTGGCCCAGCCCCTTCGGGCCGCCCTCACGGGG
G
1320 440
AGCCTGGAGACCCCGGGGCTTTTTGAGATCCTGGCCCTCCTCGGGAAGGAGCGGGCGCTCAGGCGCTTGGAGCGGGCCCTCGCCTAGGGGGTATACTGGACGCCATGAGACGGCTTCTCG S L E T P G L F E I L A L L G K E R A L R R L E R A L A *
1440 468
TCGTCCTTCTCGCCCTTTTGGGCCTCGGCCTCGGCCCAAGGCGCCATCGGGATGCGCTTCGGCTACGGGGAAGGCCTCGGCCTCACCTTCGGGGCCGGGATGGAGAACCGCCTACGGCAG
1560
AACCTCTCGGGCCGCCTCGCCGCCGACCTCGGGCCCGAGGGCCCCGGCCTGGCCCTGGAGGCCCTCCTCCTCTTCAAGCCCGACCTGGGGCAGTACGAGGGCGCCCTCAAAGGCTCCTCC
1680
CCTACGTGGCCGG
1693
L
L
R
R
A
E
F
Q
E
P
E
E
W
K
T
A
E
R
A
Y
A
L
L
F
E
T
A
E
L
D
L
Y
R
P
G
V
F
S
A
E
A
K
E
A
K
Q
G
R
V
K
K
L
L
E
G
E
Q
G
V
L
A
P
Q
L
P
L
L
K
R
E
A
L
A
Y
L
P
T
Sma I
Fig. 3. Nucleotide sequenceof the T.thermophilusgltX gene and the deduced amino acid sequence of Glu-tRNA synthetase. The underlined amino acid scquence was determined by E d m a n degradation and amino acid sequencing. The putative Shine-Dalgarno sequence is indicated.
preferential codon usage in genes from bacteria of the genus Ti~ernius[19, 26- 291. This 26-nucleotide probe was labeled with 32P, and used for Southern hybridization to T. thermophilus chromosomal DNA digested with several restriction enzymes, resulting in marked hybridization to a Sac1 fragment of about 0.95 kbp (data not shown). This 0.95-kbp Sac1 fragment was cloned into the SacI site of plasmid pUCl19 by colony hybridization. The insert was sequenced and found to include the 5'-terminal300 nucleotides of the T. thcrrnophilus g l M gene (described below). In order to clone the full-length DNA of the T. thermophilus gltX gene, the 0.95-kbp insert was labeled by nick-translation and used for Southern hybridization, yielding a single band of a 5.2-kbp KpriI digest. The 5.2-kbp KpnI fragment was cloned into plasmid pUCl18 by colony hybridization. The complete DNA sequence of a 2.4-kbp region of the KpnI fragment was determined on both strands by a sequencing strategy shown in Fig. 2, and the T. thermophilus gltX gene was found to be included in this sequenced region. The nucleotide sequence of the T. therniophilusgltXgene with its flanking regions is shown in Fig. 3.
Amino acid sequence of T. thermophilus Glu-tRNA synthetase
A nucleotide sequence coding for the amino acid sequence of the N-terminal 40 residues of T. thermophilus Glu-tRNA synthetase was found as underlined in Fig. 3. Thus, the open reading frame of the gltX gene was identified to be composed of 1407 bp, coding for 468 amino acid residues as shown in Fig. 3. The relative molecular mass was calculated to be 53 901, in agreement with that estimated previously by SDSi PAGE ( M I 50000) [15]. The amino acid composition of the Glu-tRNA synthetase, estimated from the amino acid sequence, is similar to that obtained for the hydrolysate of purified T. thermophilus Glu-tRNA synthetase [15]. Codon usage in the T. thermophilus gltX gene
+
The G C content at the third position of codons in the T. thermophilus gltX gene was found to be as high as 94%, which is similar to that (94%) of T. thermophilus Met-tRNA synthetuse gene [21] and those (85-96%) in other genes from bacteria of the genus Thermus [19,26- 291. Thus, codon usage
469
116 120 109 119 119
(7. t h . ) (R. me.) ( E . ca.) \B. su.) (B. st.)
349 ( R . re.\
468 ( T. 484 (R. 471 ( E . 483 ( B . 489 ( B .
th.) me.) co.) su.) st.)
Pig.4. Comparison of the amino acid sequences of the Glu-tRNA synthetases from T. therrnophifus, R. rneliloti, E. cofi, B. subrilis and B. siearotherrnophilus. Amino acid residua conserved in morc than three Glu-tRNA synthetases are enclosed by boxes. Identical residues to those of T. thermophilus Glu-tRNA synthetases are also encloscd. Signature sequence and similar sequence to KMSK are underlined. The abbreviations are as follows: T . th., T. thermophilus; R. me., R.meliloti; E. co., E. coli; B. su., B. subtilis; B. st., B. stearothrnnopltilus.
in the T. thermophilus &X' gene is quite different from that in the E. coligltX gene [7]. In particular, codons CGG (arginine), AAG (lysine); GAG (glutamate) are frequently used in the T. thermophilus gltX gene (Fig. 3), while these codons are rarely used in E. coli genes [30, 311. This frequent use of minor codons in the T. therrnophilus gltX gene would be a problcm in overexpression of the gltX gene in E. coli cells.
Amino-acid-sequence homology of Glu-tRNA synthetases from T. thermophilus, Rhizobium rneliloti, E. coli, B. subtilis and B. stearothermophilus Amino acid sequences of T. therrnophilus (this work), E. coli [7], R. rneliloti [32], B. suhtilis and B. steurothermophilus
[lo] Glu-tRNA synthetases are well aligned on the basis of homology (Fig. 4), indicating a structural similarity between these Glu-tRNA synthetases. Comparison of the entire amino acid sequences of T. thermophilus Glu-tRNA synthetases with those of E. coli, R. meliloti, B. subtilis and B. stewothermophilus (Fig. 4) reveals amino acid identities of 37%, 45%, 35% and 41 %, respectively and similarities in conservative replacements of 12%, 13%, 8% and 11%, respectively. Thus, T. thermophilus Glu-tRNA synthetase is the most homologous of all species tested to R . rneliloti Glu-tRNA synthetase, possibly because the R. melilotigltXgene [32] has as high a G C content as that of the T. thermophilus gltX gene. In the five Glu-tRNA synthetases, the N-terminal halves show greater similarity than the C-terminal halves (Fig. 4). As
+
470 for T. thermophilus Met-tRNA synthetase also, the N-terminal domain which is involved in the catalysis of aminoacylation shows greater similarity with other Met-tRNA synthetases than the second domain which is involved in tRNA'" recognition. Furthermore, the N-terminal halves of the Glu-tRNA synthetases show amino acid sequence similarity with the E. coli Gln-tRNA synthetase N-terminal half [33], which, by crystallographic analyses, has been shown to interact with ATP and the 3'-terminus of tRNA"'" [6]. Thus, class-I aminoacyl-tRNA synthetases [34], including Met-tRNA synthetase, Gln-tRNA synthetase and Glu-tRNA synthetase are suggested to have at least two functional domains, that is, the N-terminal domain for aminoacylation catalysis and the C-terminal domain for tRNA recognition. Putative ATP-binding site of T. thermophilus Glu-tRNA synthetase
T. thermophilus Glu-tRNA synthetase has a sequence HVG in positions 15-17, which is highly conserved in all the four Glu-tRNA synthetases (Fig. 4). Around the HVG sequence, high similarities are found among the five GlutRNA synthetases. These regions constitute the signature sequences found in several aminoacyl-tRNA synthetases which have consensus HIGH sequences essential for ATP binding [35]. It is interesting to note that the amino acid residue next to the HVG sequence is not conserved among the Glu-tRNA synthetases, unlike other aminoacyl-tRNA synthetases. Putative amino-acid-binding site
We have previously shown that the fluorescence intensity of T. thermophilus Glu-tRNA synthetase is remarkably reduced when the synthetase is bound to L-glutamate [3], suggesting the presence of a tryptophan residue near the amino-acid-binding site. In the N-terminal halves of all the sequenced Glu-tRNA synthetases which were suggested to be involved in aminoacylation catalysis, there is only one tryptophan residue (Trp62 of the T. thermophilus enzyme) conserved among all the five Glu-tRNA synthetases (Fig. 4). Furthermore, a short amino acid segment around this tryptophan is also conserved in Gln-tRNA synthetase from E. coli [33]. Thus, Trp62 of T. thermophilus Glu-tRNA synthetase may be involved in the interaction with L-glutamate. Putative binding site for tRNAG1"
T. thermophilus Glu-tRNA synthetase aminoacylates tRNA"'" from E. coli (Knl0.60 pM) as well as tRNA"" from T. thermophilus ( K , 0.65 pM) in vitro [15]. Thus, T. thermophilus and E. coli Glu-tRNA synthetases have similar tertiary structures for tRNA recognition. Aminoacyl-tRNA synthetases belonging to class I [34] have a highly conserved amino acid sequence whose consensus is KMSKS. This sequence is probably involved in the binding of the 3'-terminus of the cognate tRNA species [36, 371. T. thermophilus GlutRNA synthetase also has a similar sequence KISKR (amino acids 243 - 247), which is well conserved in all five Glu-tRNA synthetases (Fig. 4). In the case of Met-tRNA synthetases from E. coli and T. thermophilus, KMSKS motifs are located near the boundaries of the N-terminal catalytic domain and the second tRNArecognition domain [21]. Thus, like in Met-tRNA synthetases, the sequence KLSKR in Glu-tRNA synthetase is supposed to lie on the boundary of the two functional domains. In the
Fig. 5. SDS/PAGE of T.thermophilus Glu-tRNA synthetase produced in E. coli-carrying recombinant plasmids. Lane 1, relative molecular mass standards (66000, bovine serum albumin; 45000, egg albumin; 36000, glyceraldehyde-3-phosphatedehydrogenase; 29000, carbonic anhydrase; 24000, trypsinogen; 20000, trypsin inhibitor; 14000, z-lactalbumin); lane 2, heat-treated extract of E. coli cells carrying pUC118/gltX1.9; lane 3, pUC119/gltX1.9; lane 4, pEXPIJlgltX1.9; lane 5, pEXP7/gltX1.8. T. thermophilus Glu-tRNA synthetase is indicated by an arrow.
putative C-terminal domain, there are hghly conserved amino acid residues among all the sequenced Glu-tRNA synthetases. In the T. thermophilus Glu-tRNA synthetase sequence, the amino acid regions 259-266 and 305-314 are possibly involved in specific interaction with tRNAG'". In this context, we have previously found that the fluorescence intensity of T . thermophilus Glu-tRNA synthetase at 350 nm is reduced to 75% when the Glu-tRNA synthetase binds to T. thermophilus or E. coli tRNAG'" (31, suggesting the presence of a tryptophan residue near the tRNA-binding site. In fact, in the putative Cterminal domain, only one tryptophan residue (Trp312 in the T. thermophilus enzyme) is conserved among all the five GlutRNA synthetases (Fig. 4). Substitutions of Trp312 have been shown to reduce the aminoacylation activity of the Glu-tRNA synthetase (unpublished results). Furthermore, from a I3CNMR analysis on l3C-labe1ed tRNAG1",we have found that an aromatic amino acid residue of T. thermophilus Glu-tRNA synthetase is involved in interaction with the first nucleotide of the anticodon of tRNA"'" (unpublished result). Thus, we propose that the conserved tryptophan residue (Trp312 of T. thermophilus Glu-tRNA synthetase) is involved in recognition of the anticodon region of tRNAG'". Expression of the T. thermophilusgltX gene in E. coli
In order to elucidate the structure/function relationship of the thermostable Glu-tRNA synthetase by physicochemical techniques, a system for large-scale preparation of the enzyme should be established. Therefore, to overproduce the GlutRNA synthetase in E. coli, the gltX gene was inserted into various expression vectors (Fig. 1). E. coli cells transformed with pUC118/gltX1.9 and pEXP7/gltX3.9 produced thermostable T. thermophilus GlutRNA synthetase (Fig. 5, lanes 2 and 4), while E. coli cells transformed with pUC119/gltX1.9 did not (Fig. 5, lane 3). This indicates that, in the former cases, the T. thermophilus gltX gene was expressed from the lac and tac promoter in the presence of lac inducer. In SDS/PAGE, the extract of the pUCl18/gltX1.9 transformant showed one more band, the molecular mass of which was slightly higher than that of T. thermophilus Glu-tRNA synthetase. This thermostable product appears to be a fusion protein of the N-terminal region of j?-lactamase with T. therrnophilus Glu-tRNA synthetase. Furthermore, we deleted 68 nucleotides upstream of the initiation codon of the gltX gene on pEXPTJ/gltXI.9 so that the
Fig.6. The 12% S D S / P A W monitoring the purification and crystalllxation of 7. thrvmophilus C.lu-tRNA synthetase produced in E. colicarrjing pEXP7/gltXI.S. Lanl: I. ccll f r e lysatc; lane 2. lysatc after heat treatment at 70 C for 20 min: lane 3. T . rhcrmnphilus GlutRNA synthetasc purified by thrcc-step column chromatography (sce Materials and Methods); lane 4, a single-washcd crystal. The band of T.r/iermophilua (ilu-t R K A synthetase is indicatcd by :in arrow.
klg.7. Microphotograph O f crystals O f T. thermophiius GIU-tRYA synthetase. The scale bar reprcscnts 0.1 mm.
glrX mRNA was translated with the effective E. coli ShincDalgarno scqucncc (pEXP7/gltX1.8; Fig. 1). Thus. T.ther)nophilus Glu-tKNA synthetase was cfficiently overproduced in E . coli (Fig. 5. lane 5).
Purification of T. thermophilus Glu-tRNA synthetase overproduced in E. coli cells A heat-trcated extract of E. coli cclls, carrying pEXP7I gltX1.8. showcd one major band corresponding to T . rhcrrnophilus Glu-tRNA synthetasc on SDS/PAGE (Fig. 6, lane 2). Thus. heat-treatment is shown to be a useful step for purification of T. thermophilus Glu-t RNA synthctasc from I:. coli to hornogencity, as in the case of T. f h e r ~ n o p h i l uMet~ tRNA synthetase 121). We then purified 7'. rherntophilus Glut R N A synthetase from the E. coli ccll extract by chromatography on columns of DEAE-Sephacel. MonoQ and phenylSuperose, according to Materials and Mcthods (Fig. 6, lane 3). Thus, we obtained 50 mg pure T. ~hrrniophilusGlu-tKNA synthetase from 180 g E. roli cells. On SL)S,'PAGE, the mobility of the purified 7.. thrrmophilus Glu-tRNA synthetase, overproduced in E. coli cclls, was equal to that of the Glut RNA synthetase purified from 7'. thermophilus cclls (data not shown). Furthermore. the purified T . tlicvniophilus Glu-tKNA synthetase overproduced in E. c o l i w x B S active as the cn7yme from T. ~hzrinoppldu.~ cells in aruinoacylation of tRNAG'" species from E. c d i and T. fhermophihts (data not shown). Thus, the thermostable Glu-tRNA synthetase overproduced in E. co/i will be uscful for physicochcmical studies by NMK speclroscopy and X-ray crystallography.
Crystallimtion of T. thermophilus Glu-tRNA synthetase Crystalli7ation was carried out at 1 2 ' C under various conditions: the compo4tion of the precipitant 125 - 53% saturated ammonium sullate and 11 - 22% poly(ethy1ene glycol) 6000 and poly(cthylenc glycol) 30001. thc composition and pH of thc buffcr (SO mM 'L'ris,'acetate for pH 7.0 - 7.8.40 mM potassium phosphate for pH 6.5 - 7.5, SO mM Mes for pH 6.0 - 7.0, and SO mM Bistris, 50 m M Bistrisjpropane and 50 mM Pipes l o r pH 6.5) and the concentration of T. rhermophilrrs Glu-t RNA synthetase (2.6 - 10 mg at thc starting point) wcrc varied. In all trials, 2 mM MgClz and 10 mM 2-mercaptoethanol were added. Crystallization with ammonium sulfate and poly(ethylene glycol) 3000 uas unsuc-
Fig. 8. Precesion photographs of (Okl) reciprocal lattice zone. The precession anglc was 15 ' and exposure time was 24 h.
cessful. Ncedlc-like crystals. which had a strong tendency for forming a cluster. were grown from 15 - 18% poly(eth).lt.ne glycol) 6000 ;it pH 6.0-6.6. At highcr concentrations of poly(ethy1ene glyco1)WOO ( 1 9 - - 22%) or at higher pH (6.8 7.8), thinner crystals or an amorphous precipitate appeared. To improve the crystal characteristics. 2-met hylpentane-2.4diol was added to a final concentration of 1 - 6%. according to the observation of Yaremchuk et al. 1131. The butfer used (Mes, Bistris. Bistris/propanc and Pipes) slightly affected the crystal morphology. Best single crystals with dimensions 01' 0 . 1 5 x 0 . 1 5 x l . O m m (Fig. 7) appeared within 2 - 3 weeks at 12°C in a solution containing 5 mg/ml protein. 15. - 1 7 O h poly(cttiylene glyc01)6000, 50 mM Mes. pH 6.2 -6.5. 2 m M MpCIZ. 1 0 m M 2-mercaptoethanol, 1 m,M NaN3 and 1'70 2niethylpentane-2,4-diol. A crystal was washed and subjected t o SDSjPAGE. One major band wits observed with :t mobility corresponding to the intact T. !hcrrnophilus Glu-tRNA synthetase (Fig. 6, lane 4).
Preliminary crystallographic data Crystal data were obtained from precession photographs showing reflections to about 0.25 nni resolution (Fig. 8). The Okl and hol p h e s indicate mmm symmetry and an extinction law of / r = 2 n . k = 2 n. I = 2 n. I t was thus concluded that the
472 crystals belong to the orthorhombic space group P212121 with unit-cell parameters of a = 8.64 nm, b = 8.86 nm and c = 8.49 nm. The unit-cell volume is 6.50 x 10' nm3 and assumed to contain one molecule in an asymmetric unit. The value [38i is therefore to be 3010 nm3/Da>indicating that the crystals have a rather high solvent content. From measurements with a precession camera and DIPloo, the crysta1s were found to diffract to at least O.35-nm resolution. The crystals are stable against X-ray radiation and suitable for X-ray structural analysis. To obtain even better crystals of T. thermophilus Glu-tRNA synthetase and also cocrystals of Glu-tRNA synthetase and tRNAG'", further trials are now in progress.
v,
The authors are grateful to Prof. Akinori Suzuki of the Department of Agricultural Chemistry, University of Tokyo for amino acid sequencing, to Dr Susumu Nishimura of the National Cancer Center Research Institute for the synthesis of the oligodeoxyribonucleotide probe and to Dr Hiroyuki Adachi of the Department of Agricultural Chemistry. University of Tokyo for his helpful discussion. This work was supportcd in part by Grant-in-Aid for Specially Promoted Research (60060004) and Grants-in-Aid for Scientific Research on Priority Areas (01656002 and 02238102) from the Ministry of Education, Science and culture of Japan, by a Bioscience Grant for International Joint Research Project from the New Energy and Jndustrial Technology Devclopment Organization, and by a Research Grant from International Human Frontier Science Program Organization.
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