tRNA Synthetase of Bacillus subtilis - Semantic Scholar
Vol. 267, No. 13, Issue of May 5, pp. 9146-9149,1992 Printed in lI S.A.
CHEMISTRY THEJOURNAL OF BIOLOGICAL
0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.
Mutational Identification of an Essential Tryptophan in TryptophanyltRNA Synthetase of Bacillus subtilis" (Received for publication, December 9, 1991)
King-Chuen Chow$, HongXueO, Wen Shi, and J. Tze-Fei WongV From the Department of Biochemistry, University of Toronto, Toronto, Canada M5S lA8
The strongly conserved single tryptophan residue, Trp"', in Bacillus subtilis tryptophanyl-tRNA synthetase has been mutagenized via sitedirection singly into Gln, Ala, and Phe. All three mutant enzymes were inactive toward the catalysis of tRNA tryptophanylation. The Trp" + Phe mutant has been subcloned into the high expression plasmid pKK223-3 to yield the recombinant plasmid pKSW-F92. Growth of bacteria carrying the latter plasmid made possible the purification of the mutant TrpRS-F92 enzyme to homogeneity. This mutant enzyme was deficient in ultraviolet absorbance and fluorescence relative to the wild type enzyme and inactive in the partial reaction of Trpactivation as well as the overall reaction of tRNA tryptophanylation. Furthermore, unlike the wild type B. subtilis trpS gene, the mutant trpS-Fg2 gene upon transformation into Escherichia colitrpS 10343 failed to complement the temperaturesensitive trpS mutation of the host cells. Trpg2therefore represents an essential residue both in vitroand in vivofor the function of the tryptophanyl-tRNA synthetase.
MATERIALS ANDMETHODS
Bacteria and Plasmids-Escherichia coli JM109 (5) was employed as host cell for expressing the B. subtilis trpS gene. The bacterial culture was grown at 37 "C in LB medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl) supplemented with 75 pg/ml ampicillin. E. coli trpF 10343, a tryptophan auxotroph containinga temperature-sensitive trpS gene (6), could grow in M9-glucose minimal medium at 32 "C but not at 42 "C. It thus furnished a basis for testing in vivo complementation to the E.coli ts trpS gene in this medium. The trpSgene of B. subtilis encoding wild type TrpRS was carried in the pUC8-derived pTSQ2 plasmid (1). The plasmid pKK223-3, containing the highlyefficienttac promotor (7), was used as the cloning vector to overexpress either thewild type or the mutated trpS gene. Site-directed Mutagenesis and Expression of trpS Gene-Synthetic oligonucleotides 18 nucleotides in length were used to convert the codon for Trpg2on the B.subtilis gene for TrpRS to thatof Gln, Ala, or Phe. The mutagenesis procedure used in this study was based on (8).Since an initial cloning that developed byNakamaye and Eckstein of the trpS-carrying fragment from pTSQ2 into pEMBL8 (9)gave a low yield of single-stranded DNA, the gene was cloned instead into the M13 phage. The mutated M13 phage DNAs were used to transform E. coli JM109, and plaque-purified phages were employed for the preparation of single-stranded DNA.After the presence of a desired mutation within the trpS gene had been confirmed by sequencing theregion of interest, the nucleotide sequence of the entire gene was determinedtoensurethe absence of anyadventitious The accuracy of protein synthesis is essential to thesurvival mutations. The yield of mutations with this method was 80-90%. of any organism, and thefidelity of aminoacyl-tRNA synthe- Since the trpS gene for unidentified reasons was not significantly expressed in the M13 phage system, the mutated trpS-carrying fragtases represents a key requirement in thisregard. Insight into ment was recloned into pUC8 and propagated in JM109 for expresthe structure-function relationship of aminoacyl-tRNA syn- sion. One of the three mutant genes, which encodes Phe at position thetases is therefore a subject of intensive experimental in- 92, was further subcloned into thehigh expression plasmid pKK2233 (from Pharmacia LKB Biotechnology Inc.) to yield pKSW-F92 for vestigation. overproduction of the gene product, as had been performed previously Tryptophanyl-tRNA synthetase (TrpRS),' a dimeric en- for the wild type trpS gene yielding the pKSWl plasmid (10). zyme, contains the smallest subunit chains among known The over 8000-fold purification of native TrpRS from B. subtilis aminoacyl-tRNA synthetases. Therefore itprovides a partic- required a combination of four purification steps includingtwo high of the ularly valuable system for structure-function analysis through performance liquid chromatography steps (11). On account high expression from E. coli JM109 cells bearing either pKSWl or mutagenesis. Of the prokaryotic/organelle TrpRS sequences pKSW-F92, only a 2-fold purification employing chromatography on that havebeen determined, that of Bacillus subtilis (1) is DEAE-Sephacel and hydroxylapatite sufficed to yield a homogeneous exceptional in that it contains onlyasingle tryptophanyl enzyme? Enzyme Assays-The catalyzed tryptophanylation of tRNA was residue at Trpg2.In contrast, there are 2 Trp residues in the assayed based on the formationof ['HITrp-tRNA, and the ATP-PPi Escherichia coli enzyme (2), 3 in the Bacillus stearothermoexchange reaction was assayed based on the incorporation of["PI philus enzyme (3), and 4 in the yeast mitochondrial enzyme pyrophosphate into ATP, both as described (11).Trp-hydroxamate (4). Accordingly the object of this study is to apply siteformation was determined in accordance with Davie (12). Protein concentrations were determined using the bicinchoninic directed mutagenesis to the B. subtilis enzyme in order to acid (BCA) protein assay reagent from Pierce Chemical Co. (13). define thepossible role of Trpg2. Ultraviolet absorption spectra were determined with a Kontron Uvi* This study was supported by the Medical Research Council of kon 810 spectrophotometer, and fluorescence spectra with a Spex Canada. The costsof publication of this article were defrayed in part Fluorolog fluorometer. be by the paymentof page charges.This article must therefore hereby RESULTS marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Actiuities of Mutated Enzymes-When the three mutated $ Recipient of a British Commonwealth Scholarship. trpS genes cloned into pUC8 were expressed in JM109, and § Recipient of a University of Toronto Open Fellowship. 1 Present address: Dept. of Biochemistry, Hong Kong University H. Xue, T. A. Webster, X. F. Wu, and J. T. Wong, unpublished of Science and Technology, Clear Water Bay, HongKong. observations. The abbreviationused is: TrpRS, tryptophyl-tRNA synthetase.
9146
Mutation of TrpRS
9147
the enzyme extracts assayed for tRNA tryptophanylation, all three of the mutants, bearing Alag2,Glnsl, or Pheg2, respecO.*r tively, displayeda practically complete loss of activity relative to the wild type enzyme (Table I). 0.6 Subcloning of the Trpg2Phe Mutant Gene-While the tryptophanylation assaywas readily measuredon enzyme extracts prepared from JM109 cells carrying the pTSQ2plasmid, the fluorescence, ATP-PPi exchange, and Trp-hydroxamate formation studies required a purified enzyme. For this purpose, a high-level expression of the mutant TrpRS-F92 enzyme had t o be sought utilizing the same system thatyielded the high level expression plasmid pKSWl for wild type TrpRS. Accordingly, as in the construction of pKSW1, the trpS-bearing 1.49-kilobase BamHI-EcoRI fragment derived from pTSQ2 (1) was cloned into phage M13, and mutagenized to generate 250 300 a n EcoRI site flanking the very 5' end of the trpS gene. WAVELENGTH (nm) Additionally, the TGGcodon for Trpgzwas converted toTTT, encoding for phenylalanine, before the mutated genewas FIG. 2. Ultraviolet absorption spectra.Upper curve, wild type cleaved by EcoRI and inserted into the EcoRI site of pKK223- TrpRS; lower curve, TrpRSF92. Both samples contained 0.73 mg/ 3 and transformed into JM109. The mutant trpS gene re- ml protein. covered from the transformants coulddisplay one of two possible orientations withrespect to thetac promotor on the vector. Atransformant that carried the plasmid with thetrpS gene aligned downstream to thetac promotor was designated pKSW-F92. Uponexpression of the mutant trpS gene on pKSW-F92 in JM109, the yield of the mutant TrpRS-F92 enzyme was comparable with that of the wild type enzyme expressed from the pKSWl plasmid, exceeding 50% of total proteins in the crude extract (10). Purification of the TrpRSF92 so obtained readily yielded the homogeneousenzyme shown in Fig. 1. Characterization of theTrpRS-F92 Enzyme-Compared .....,.................. *........+ .........,.... .'.,.""."'. 0 with wild type TrpRS at the same protein concentration as 290 WAVELENGTH 345 (nm) 400
C K
/_."""_ "_
'
""" """""""
.........................
TABLE I Tvptophnnylation of tRNA by extracts from E. coli JM109 cells cloned with the wild type and three mutantgenes for TrpRS Enzvme
Relative activitv
Wild type Trp"' + Ala Trps' + Gln Tw,"* + Phe
100
g
l
.
o
,
,
,
,
,
t
#
~
l
0.135 mg/ml ---F92 0.135 mp/ml "wT
%
.- chondria contain 3, 2, and 4 Trp residues, respectively, only kk that Trp residue on these molecules which corresponds to La conservation Trpg2 in B. subtilis is conserved.Sequence Ia around this residue, which extends to all four enzymes (Fig. -0 I 2 3 4 7), agrees with the conserved Trp fulfilling a central role in Trp RS (nM) the catalytic mechanisms of this family of aminoacyl-tRNA FIG. 5. ATP-PPi exchange reaction. Uppercurue, wild type synthetases. TrpRS; lower curue, TrpRS-F92. The exact role of Trpg2 remains to be determined. Since Trp isone of themost hydrophobic of amino acids,one TABLEI1 possibility might be that Trpg2 interacts with the tryptophan, Trp-hydroxamate formationby wild type TrpRS and the TrpRS-F92 ATP, or tRNA substrate of the enzyme through hydrophobic mutant enzyme or stacking interactions. Previously, it has been found that Enzyme Relative activity substrate hydrophobicity among the fluorotryptophans is a % key factor determining theselectivity of TrpRS toward these Wild type 100 substrates (11).Recently, 2 Trp residues on a single-stranded 0.8 TmS2+ Phe DNA-binding protein also have been found to be important in complex formation with DNA through base stacking (15). Because Trpg2is required for the formation of Trp-AMP in t o be expected from a loss of Trpg2 from the latter. of Trp-tRNA, Enzymatically, the deficient esterification of tryptophan to the absenceof tRNA noless than the formation tRNA was as evident with the purified TrpRS-F92 enzyme its role cannot be restricted to oneof interaction with tRNA (Fig. 4) as with its enzyme extract (Table I). In both instancesalone. Being the only tryptophan residue on the synthetase molthe mutant enzyme exhibited little observable activity. by absorption, Since the TrpRS of B. subtilis catalyzes a Trp-dependent ecule, Trpg2 is particularly open to observation fluorescence, and NMR spectroscopies. Application of these ATP-PPi exchange reaction ( l l ) , t h eformation of Trp-tRNA andother physical measurements will undoubtedly throw evidently proceeds through a Trp-AMPintermediateas further light on this crucial amino acid residueon the smallest shown below. 0
100
200 300 Trp RS (PM)
400
500
/i
+ ATP -+ Trp-AMP + PPI Step 2: Trp-AMP + tRNA + Trp-tRNA + AMP Step 1: Trp
The ATP-PPiexchange reaction depends on Step while 1, the FIG. 6. Positions of Trp residues on tryptophanyl-tRNA tryptophanylation of tRNA depends on both Steps 1 and 2. synthetases. T h e loss of tRNA tryptophanylation by TrpRS-F92 could result from a loss of Step 1 or Step 2 or both. T o assess the Bsubt Gln Ser Glu Val Pro Ala His Ala Gln Ala90 Gln Ser Glu Val Pro Ala His Ala Gln Ala89 B.sfear ability of TrpRS-F92 to catalyze Step 1, the ATP-PPi exGln Ser HIS Val Pro Glu His Ala Gln Leu91 E.co1i Y.mito Gln Ser Ala Ile Pro Gln His Ser Glu Leu127 change reaction was measured according to Xu et al. (11). B.subf Gly Trp Met Met Gln Cys Val Ala Tyr llel00 Bsfear Ala Trp Met Leu Gln Cys Ile Val Tyr Ne99 T h e F92 enzyme was found to be severely defective relative E.coli Gly Trp Ala Leu Asn C s Tyr Thr Tyr PhelOl Y.mito His Trp Leu Leu Ser T& Leu Ala Ser Met137 to thewild type (Fig. 5). B.subf Another method for assessing the formation of Trp-AMP Gly Glu Leu Glu Arg Met Thr Gln Phe Lys110 Bsfear Gly Glu Leu Glu Arg Met Thr Gln Phe LyslO9 €.coli Gly Glu Leu Ser Arg Met Thr Gln Phe L y s l l l in Step 1 was to monitor the reaction of this intermediate Y.mito Gly Leu Leu Asn Arg Met Thr Gln Trp Lys147 with hydroxylamine to produce Trp-hydroxamate. On this 5.subl Asp Lys Ser113 GI” Lys Serl12 5.sfear basis, TrpRS-F92 was again found to be deficient, showing E.wB Asp Lys Serl14 Y.mito Ser Lys 9 1 1 5 0 less than 1%of wild type activity (Table 11). Previously it was observed that the cloned wild type B. FIG. 7. Sequence conservation around the unique Trp resisubtilis trpS gene on pKSWl could complement the temper- due on B. subtilis tryptophanyl-tRNA synthetase.
Mutation of TrpRS of polypeptide chains among the aminoacyl-tRNA synthetases. Acknowledgment-We are indebted to Dr. J. Friesen for preparation of oligonucleotides. REFERENCES 1. Chow, K. C., and Wong, J. T. (1988) Gene (Amst.) 73,537-543 2. Hall, C. V., Van Cleemput, M., Muench, K. H., and Yanofsky, C. (1982) J. Biol. Chem. 257,6132-6136 3. Barstow, D. A., Sharman, A. F., Atkinson, T., and Minton, N. P. (1986) Gene (Amst.) 46,37-45 4. Myers, A. M., and Tzagoloff, A. (1985) J. Biol.Chem. 260, 15371-15377 5. Yanish-Perron, C., Vieira, J., and Messing, J. (1985) Gene (Amst.) 33,103-199 6. Doolittle, W . F., and Yanofsky, C. (1968) J. Bacteriol. 95, 12831294
9149
7. Brosius, J., and Holy, A. (1984) Proc. Natl. Acad. Sci. U. S. A . 8 1,6929-6933 8. Nakamaye, K. L., and Eckstein, F. (1986) Nucleic Acids Res. 1 4 , 9679-9698 9. Dente, L., Cesareni, G., and Cortese, R. (1983) Nucleic Acids Res. 11,1645-1655 10. Shi, W . , Chow, K. C., and Wong, J. T. (1989) Biochern. Cell Biol. 68,492-495 11. Xu, Z. J., Love, M. L., Ma, L. Y. Y., Blum, M., Bronskill, P. M., Bernstein, J., Grey, A. A., Hofmann, T., Camerman, N., and Wong, J. T. (1989) J. Biol. Chern. 264,4304-4311 12. Davie, E. W . (1962) Methods Enzyrnol. 5 , 718-722 13. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Anal. Biochern. 150, 76-85 14. Lakowicz, J. R. (1983) Principles of Fluorescence Spectroscopy, pp, 342-368, Plenum Press, New York 15. Khamis, M. I., Casas-Finet, J. R., and Maki, A. H. (1987) J . Biol. Chem. 262,10938-10945
CHEMISTRY THEJOURNAL OF BIOLOGICAL
0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.
Mutational Identification of an Essential Tryptophan in TryptophanyltRNA Synthetase of Bacillus subtilis" (Received for publication, December 9, 1991)
King-Chuen Chow$, HongXueO, Wen Shi, and J. Tze-Fei WongV From the Department of Biochemistry, University of Toronto, Toronto, Canada M5S lA8
The strongly conserved single tryptophan residue, Trp"', in Bacillus subtilis tryptophanyl-tRNA synthetase has been mutagenized via sitedirection singly into Gln, Ala, and Phe. All three mutant enzymes were inactive toward the catalysis of tRNA tryptophanylation. The Trp" + Phe mutant has been subcloned into the high expression plasmid pKK223-3 to yield the recombinant plasmid pKSW-F92. Growth of bacteria carrying the latter plasmid made possible the purification of the mutant TrpRS-F92 enzyme to homogeneity. This mutant enzyme was deficient in ultraviolet absorbance and fluorescence relative to the wild type enzyme and inactive in the partial reaction of Trpactivation as well as the overall reaction of tRNA tryptophanylation. Furthermore, unlike the wild type B. subtilis trpS gene, the mutant trpS-Fg2 gene upon transformation into Escherichia colitrpS 10343 failed to complement the temperaturesensitive trpS mutation of the host cells. Trpg2therefore represents an essential residue both in vitroand in vivofor the function of the tryptophanyl-tRNA synthetase.
MATERIALS ANDMETHODS
Bacteria and Plasmids-Escherichia coli JM109 (5) was employed as host cell for expressing the B. subtilis trpS gene. The bacterial culture was grown at 37 "C in LB medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl) supplemented with 75 pg/ml ampicillin. E. coli trpF 10343, a tryptophan auxotroph containinga temperature-sensitive trpS gene (6), could grow in M9-glucose minimal medium at 32 "C but not at 42 "C. It thus furnished a basis for testing in vivo complementation to the E.coli ts trpS gene in this medium. The trpSgene of B. subtilis encoding wild type TrpRS was carried in the pUC8-derived pTSQ2 plasmid (1). The plasmid pKK223-3, containing the highlyefficienttac promotor (7), was used as the cloning vector to overexpress either thewild type or the mutated trpS gene. Site-directed Mutagenesis and Expression of trpS Gene-Synthetic oligonucleotides 18 nucleotides in length were used to convert the codon for Trpg2on the B.subtilis gene for TrpRS to thatof Gln, Ala, or Phe. The mutagenesis procedure used in this study was based on (8).Since an initial cloning that developed byNakamaye and Eckstein of the trpS-carrying fragment from pTSQ2 into pEMBL8 (9)gave a low yield of single-stranded DNA, the gene was cloned instead into the M13 phage. The mutated M13 phage DNAs were used to transform E. coli JM109, and plaque-purified phages were employed for the preparation of single-stranded DNA.After the presence of a desired mutation within the trpS gene had been confirmed by sequencing theregion of interest, the nucleotide sequence of the entire gene was determinedtoensurethe absence of anyadventitious The accuracy of protein synthesis is essential to thesurvival mutations. The yield of mutations with this method was 80-90%. of any organism, and thefidelity of aminoacyl-tRNA synthe- Since the trpS gene for unidentified reasons was not significantly expressed in the M13 phage system, the mutated trpS-carrying fragtases represents a key requirement in thisregard. Insight into ment was recloned into pUC8 and propagated in JM109 for expresthe structure-function relationship of aminoacyl-tRNA syn- sion. One of the three mutant genes, which encodes Phe at position thetases is therefore a subject of intensive experimental in- 92, was further subcloned into thehigh expression plasmid pKK2233 (from Pharmacia LKB Biotechnology Inc.) to yield pKSW-F92 for vestigation. overproduction of the gene product, as had been performed previously Tryptophanyl-tRNA synthetase (TrpRS),' a dimeric en- for the wild type trpS gene yielding the pKSWl plasmid (10). zyme, contains the smallest subunit chains among known The over 8000-fold purification of native TrpRS from B. subtilis aminoacyl-tRNA synthetases. Therefore itprovides a partic- required a combination of four purification steps includingtwo high of the ularly valuable system for structure-function analysis through performance liquid chromatography steps (11). On account high expression from E. coli JM109 cells bearing either pKSWl or mutagenesis. Of the prokaryotic/organelle TrpRS sequences pKSW-F92, only a 2-fold purification employing chromatography on that havebeen determined, that of Bacillus subtilis (1) is DEAE-Sephacel and hydroxylapatite sufficed to yield a homogeneous exceptional in that it contains onlyasingle tryptophanyl enzyme? Enzyme Assays-The catalyzed tryptophanylation of tRNA was residue at Trpg2.In contrast, there are 2 Trp residues in the assayed based on the formationof ['HITrp-tRNA, and the ATP-PPi Escherichia coli enzyme (2), 3 in the Bacillus stearothermoexchange reaction was assayed based on the incorporation of["PI philus enzyme (3), and 4 in the yeast mitochondrial enzyme pyrophosphate into ATP, both as described (11).Trp-hydroxamate (4). Accordingly the object of this study is to apply siteformation was determined in accordance with Davie (12). Protein concentrations were determined using the bicinchoninic directed mutagenesis to the B. subtilis enzyme in order to acid (BCA) protein assay reagent from Pierce Chemical Co. (13). define thepossible role of Trpg2. Ultraviolet absorption spectra were determined with a Kontron Uvi* This study was supported by the Medical Research Council of kon 810 spectrophotometer, and fluorescence spectra with a Spex Canada. The costsof publication of this article were defrayed in part Fluorolog fluorometer. be by the paymentof page charges.This article must therefore hereby RESULTS marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Actiuities of Mutated Enzymes-When the three mutated $ Recipient of a British Commonwealth Scholarship. trpS genes cloned into pUC8 were expressed in JM109, and § Recipient of a University of Toronto Open Fellowship. 1 Present address: Dept. of Biochemistry, Hong Kong University H. Xue, T. A. Webster, X. F. Wu, and J. T. Wong, unpublished of Science and Technology, Clear Water Bay, HongKong. observations. The abbreviationused is: TrpRS, tryptophyl-tRNA synthetase.
9146
Mutation of TrpRS
9147
the enzyme extracts assayed for tRNA tryptophanylation, all three of the mutants, bearing Alag2,Glnsl, or Pheg2, respecO.*r tively, displayeda practically complete loss of activity relative to the wild type enzyme (Table I). 0.6 Subcloning of the Trpg2Phe Mutant Gene-While the tryptophanylation assaywas readily measuredon enzyme extracts prepared from JM109 cells carrying the pTSQ2plasmid, the fluorescence, ATP-PPi exchange, and Trp-hydroxamate formation studies required a purified enzyme. For this purpose, a high-level expression of the mutant TrpRS-F92 enzyme had t o be sought utilizing the same system thatyielded the high level expression plasmid pKSWl for wild type TrpRS. Accordingly, as in the construction of pKSW1, the trpS-bearing 1.49-kilobase BamHI-EcoRI fragment derived from pTSQ2 (1) was cloned into phage M13, and mutagenized to generate 250 300 a n EcoRI site flanking the very 5' end of the trpS gene. WAVELENGTH (nm) Additionally, the TGGcodon for Trpgzwas converted toTTT, encoding for phenylalanine, before the mutated genewas FIG. 2. Ultraviolet absorption spectra.Upper curve, wild type cleaved by EcoRI and inserted into the EcoRI site of pKK223- TrpRS; lower curve, TrpRSF92. Both samples contained 0.73 mg/ 3 and transformed into JM109. The mutant trpS gene re- ml protein. covered from the transformants coulddisplay one of two possible orientations withrespect to thetac promotor on the vector. Atransformant that carried the plasmid with thetrpS gene aligned downstream to thetac promotor was designated pKSW-F92. Uponexpression of the mutant trpS gene on pKSW-F92 in JM109, the yield of the mutant TrpRS-F92 enzyme was comparable with that of the wild type enzyme expressed from the pKSWl plasmid, exceeding 50% of total proteins in the crude extract (10). Purification of the TrpRSF92 so obtained readily yielded the homogeneousenzyme shown in Fig. 1. Characterization of theTrpRS-F92 Enzyme-Compared .....,.................. *........+ .........,.... .'.,.""."'. 0 with wild type TrpRS at the same protein concentration as 290 WAVELENGTH 345 (nm) 400
C K
/_."""_ "_
'
""" """""""
.........................
TABLE I Tvptophnnylation of tRNA by extracts from E. coli JM109 cells cloned with the wild type and three mutantgenes for TrpRS Enzvme
Relative activitv
Wild type Trp"' + Ala Trps' + Gln Tw,"* + Phe
100
g
l
.
o
,
,
,
,
,
t
#
~
l
0.135 mg/ml ---F92 0.135 mp/ml "wT
%
.- chondria contain 3, 2, and 4 Trp residues, respectively, only kk that Trp residue on these molecules which corresponds to La conservation Trpg2 in B. subtilis is conserved.Sequence Ia around this residue, which extends to all four enzymes (Fig. -0 I 2 3 4 7), agrees with the conserved Trp fulfilling a central role in Trp RS (nM) the catalytic mechanisms of this family of aminoacyl-tRNA FIG. 5. ATP-PPi exchange reaction. Uppercurue, wild type synthetases. TrpRS; lower curue, TrpRS-F92. The exact role of Trpg2 remains to be determined. Since Trp isone of themost hydrophobic of amino acids,one TABLEI1 possibility might be that Trpg2 interacts with the tryptophan, Trp-hydroxamate formationby wild type TrpRS and the TrpRS-F92 ATP, or tRNA substrate of the enzyme through hydrophobic mutant enzyme or stacking interactions. Previously, it has been found that Enzyme Relative activity substrate hydrophobicity among the fluorotryptophans is a % key factor determining theselectivity of TrpRS toward these Wild type 100 substrates (11).Recently, 2 Trp residues on a single-stranded 0.8 TmS2+ Phe DNA-binding protein also have been found to be important in complex formation with DNA through base stacking (15). Because Trpg2is required for the formation of Trp-AMP in t o be expected from a loss of Trpg2 from the latter. of Trp-tRNA, Enzymatically, the deficient esterification of tryptophan to the absenceof tRNA noless than the formation tRNA was as evident with the purified TrpRS-F92 enzyme its role cannot be restricted to oneof interaction with tRNA (Fig. 4) as with its enzyme extract (Table I). In both instancesalone. Being the only tryptophan residue on the synthetase molthe mutant enzyme exhibited little observable activity. by absorption, Since the TrpRS of B. subtilis catalyzes a Trp-dependent ecule, Trpg2 is particularly open to observation fluorescence, and NMR spectroscopies. Application of these ATP-PPi exchange reaction ( l l ) , t h eformation of Trp-tRNA andother physical measurements will undoubtedly throw evidently proceeds through a Trp-AMPintermediateas further light on this crucial amino acid residueon the smallest shown below. 0
100
200 300 Trp RS (PM)
400
500
/i
+ ATP -+ Trp-AMP + PPI Step 2: Trp-AMP + tRNA + Trp-tRNA + AMP Step 1: Trp
The ATP-PPiexchange reaction depends on Step while 1, the FIG. 6. Positions of Trp residues on tryptophanyl-tRNA tryptophanylation of tRNA depends on both Steps 1 and 2. synthetases. T h e loss of tRNA tryptophanylation by TrpRS-F92 could result from a loss of Step 1 or Step 2 or both. T o assess the Bsubt Gln Ser Glu Val Pro Ala His Ala Gln Ala90 Gln Ser Glu Val Pro Ala His Ala Gln Ala89 B.sfear ability of TrpRS-F92 to catalyze Step 1, the ATP-PPi exGln Ser HIS Val Pro Glu His Ala Gln Leu91 E.co1i Y.mito Gln Ser Ala Ile Pro Gln His Ser Glu Leu127 change reaction was measured according to Xu et al. (11). B.subf Gly Trp Met Met Gln Cys Val Ala Tyr llel00 Bsfear Ala Trp Met Leu Gln Cys Ile Val Tyr Ne99 T h e F92 enzyme was found to be severely defective relative E.coli Gly Trp Ala Leu Asn C s Tyr Thr Tyr PhelOl Y.mito His Trp Leu Leu Ser T& Leu Ala Ser Met137 to thewild type (Fig. 5). B.subf Another method for assessing the formation of Trp-AMP Gly Glu Leu Glu Arg Met Thr Gln Phe Lys110 Bsfear Gly Glu Leu Glu Arg Met Thr Gln Phe LyslO9 €.coli Gly Glu Leu Ser Arg Met Thr Gln Phe L y s l l l in Step 1 was to monitor the reaction of this intermediate Y.mito Gly Leu Leu Asn Arg Met Thr Gln Trp Lys147 with hydroxylamine to produce Trp-hydroxamate. On this 5.subl Asp Lys Ser113 GI” Lys Serl12 5.sfear basis, TrpRS-F92 was again found to be deficient, showing E.wB Asp Lys Serl14 Y.mito Ser Lys 9 1 1 5 0 less than 1%of wild type activity (Table 11). Previously it was observed that the cloned wild type B. FIG. 7. Sequence conservation around the unique Trp resisubtilis trpS gene on pKSWl could complement the temper- due on B. subtilis tryptophanyl-tRNA synthetase.
Mutation of TrpRS of polypeptide chains among the aminoacyl-tRNA synthetases. Acknowledgment-We are indebted to Dr. J. Friesen for preparation of oligonucleotides. REFERENCES 1. Chow, K. C., and Wong, J. T. (1988) Gene (Amst.) 73,537-543 2. Hall, C. V., Van Cleemput, M., Muench, K. H., and Yanofsky, C. (1982) J. Biol. Chem. 257,6132-6136 3. Barstow, D. A., Sharman, A. F., Atkinson, T., and Minton, N. P. (1986) Gene (Amst.) 46,37-45 4. Myers, A. M., and Tzagoloff, A. (1985) J. Biol.Chem. 260, 15371-15377 5. Yanish-Perron, C., Vieira, J., and Messing, J. (1985) Gene (Amst.) 33,103-199 6. Doolittle, W . F., and Yanofsky, C. (1968) J. Bacteriol. 95, 12831294
9149
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