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Misaminoacylation and tRNA-dependent transamidation in Streptomyces coelicolor A3(2)
FEMS Microbiology Letters 92 (1992) 19-22 © 1992 Federation of European Microbiological Societies 0378-1097/92/$(|5.00
Published by Elsevier
FEMSLE 04818
Misaminoacylation and tRNA-dependent transamidation in Streptomyces coelicolor A3(2) Astrid Sch6n ~ and J. Stefan Rokem b a Laboratorium fiir Biochemie, Unil~er.~itiit Bayreuth, Bayreuth, FRG and ~ Department o]'Applied Microbiology. h~stitute ~f Micmbioh~h~', Jertt~ah'm. israel Received 28 October 1991 Revision received 10January 1992 Accepted 13 January 1992
Key words: Amidotransferase; Mischarging; Glutamyl-tRNA synthetase: Protein synthesis; Streptomyces; tRNA
1. SUMMARY
Streptomyces coelicolor was found to be devoid of glutaminyl-tRNA synthetase, in this bacterium, tRNA ~" is aminoacylated by glutamyltRNA synthetase to yield glutamyl-tRNATM, followed by correction to glutaminyl-tRNAc;tn by a tRNA-dcpendent amidotransferase.
2. INTRODUCTION The formation of glutamyl-tRNAC;In (GIntRNAG~"), the activated amino acid precursor re-
Correspondence to: A, Schi~n. Laboratorium fiir Biochemie. Universit~t Bayreuth, P.O. Box 10 12 51, 8580 Bayreuth, FRG.
quired for insertion of glutamine into polypeptide chains, proceeds via a two-step mechanism in Gram-positive bacteria, Archaebacteria, mitochondria and chloroplasts [1-5]. These sources do not possess any glutaminyl-tRNA synthetase (GlnRS), but instead contain a naturally mischarging glutamyl-tRNA synthetase (GIuRS}. This enzyme misacylates tRNA Gin with glutamate to yield GIu-tRNA c;~", which is subsequently convetted to GIn-tRNAc'l" by a tRNA dependent amidotransferase [1,6-8]. The transamidation requires, similar to aminoacyI-tRNA formation, the presence of Mg 2~ and the consumption of ATP, and in addition glutamine or asparagine as an amide group donor. In this communication we describe the preparation of a cell free extract capable of tRNA dependent reactions, and we show that this mischarging pathway and the subsequent transamidation to yield GIn-tRNAt~n is also used by the Gram-positive bacterium, Strep-
tomyces coelicolor.
3. MATERIALS AND METHODS All biochemicals were of the highest purity available. [~4C]-amino acids were from Amersham Radiochemicals or New England Nuclear.
3.1. Bacterial stra#~ and growth conditions S. coelicolor A3(2) was obtained from J. Piret (Northeastern University, Boston, MA, USA). Solid medium for growth of the bacterium was R2YE [9]. Liquid cuItures were grown in AM medium [10] to the late logarithmic growth phase. 3.2. Cell extract After harvesting by centrifugation, the ceils were suspended in 2 volumes of synthetase buffer (SB; 50 mM Tris. HCI pH 7.5; 1 mM Na2EDTA; 10 mM MgCI2; 10 mM /3-mercaptoethanol; 0.2 m'M PMSI;) with the aid of a 'Potter' type tissue homogenizer with Teflon pestle. The cells were opened by N, cavitation [11], and ribosomes and cell debris were removed by centrifugation at 100000 ×g and 4°C for 1 h. Endogenous tRNAs were removed by adjusting the extract to 0.3 M NaCI and passing it over DE 52 (Whatman) equilibrated with SB containing 0.3 M NaCI [12]. The postribosomal supernatant was dialysed against 2 × 500 volumes of SB containing 0.2 mM PMSF and 44% glycerol (v/v), and stored at -20°C. The protein concentration in the cell extract was estimated spectrophotometrically according to [13]. 3.3. tRNA preparation Cells grown as above were suspended in buffer A (0.14 M NaAc, pH 4,5) and disrupted directly into saturated phenol. The aqueous phase was re-extracted with phenol and washed with chloroform/isoamylalcohol (24: 1). The combined aqueous phases were bound to a DEAE cellulose column (Whatman DE 52, 1.5 × 5 cm) [14] equilibrated with buffer A, the column was washed with buffer A containing 0.3 M NaCI, and the tRNA fraction was eluted with buffer A containing 1 M NaCI. Nucleic acids were precipitated by addition of 2 volumes of ethanol, collected by centrifugation, dried under vacuum and dissolved in HzO. Bulk tRNA concentration was estimated
spectrophotometrically, with 1.6 nmol/ml tRNA corresponding to an A z~0 of 1.
3.4. Amincacylation assays Aminoacylation assays [15] were performed in 100 mM Tris. HCI, pH 7.5; 10 mM MgCI2; 4 mM fl-mcrcaptoethanol; 5 mM ATP; 15 p.M [~4C]amino acid (leucine, 342 mCi/mmol; glutamic acid, 293 mCi/mmol; glutamine, 287 mCi/mmol), 15 ~M bulk tRNA, and 1.5 mg/ml cell extract. 10-~1 aliquots were spotted on filter paper disks that were washed in 10% TCA, 5% TCA and ethanol. Radioactivity was detected by liquid scintillation counting. 3.5. Amidotransferase assays The composition of the complete amidotransferase assay was identical to the aminoacylation and contained, in addition, 0.33 mM [~2C]glutamine. The reactions were stopped by adjusting them to pH 4.5 and 0.2 M NaAc, followed by phenol extraction and precipitation of the aqueous phases. The nucleic acids were deacylated by incubation in 50 mM NaHCO 3 (pH 9.1) at 50°C for 15 min, and the resulting hydrolysate was dried down and applied to a cellulose TLC plate (20 cm long) at 1.5 cm from the edge of the plate. The chromatogram was developed in isopropanol:HCOOH:H_,O (80:4:20; [16]) and scanned for the presence of radioactivity with a Berthoid Scanner !I (model LB 2723).
4. RESULTS AND DISCUSSION
4.1. Preparation of a cell free extract from S. coelicolor capable of aminoacylation A system was established that allowed the investigation of possible tRNA-dependent regulatory mechanisms. We have chosen tRNAaminoacylation as an assay system for RNAmetabolic reactions since it is simple to monitor. Reaction conditions were optimised with leucine and glutamic acid as [14C]-Iabelled amino acids and S. coelicolor tRNA a~ substrate. A control experiment, where exogenous bulk tRNA was omitted from the reaction mixture containing [14C]-Ieucine, gave no significant incorporation of
A
Leu ..,.,.
~s "6
E ..,=4
~2 ,~
~
Gtu
ml "
2
5
,
10
= Gin
20
iment lacking tRNA (see 4.1.), clearly demonstrate that even after a prolonged incubation time, there was no significant incorporation of [14C]-glutamine into acid-precipitable material. In contrast, aminoacylation with [~4C]-glutamie acid r~:~ultcd in an appreciable amount of this amino acid bound to tRNA. It is thus likely that S. coelicolor, like all other Gram-positive bacteria investigated so far, do not possess GlnRS, but instead make use of a naturally misacylating GluRS and a two-step pathway for the synthesis of GIn-tRNA (;~" via a misacylated Giu-tRNA G~" intermediate [1,4].
time (min) Fig. I. Time course of aminoacylalion ol S. codii'olor IRNA with a postribosomal supernatanl from S. cot'licc~h~r. Reactions were performed as described in MA'n~RI,Xl,S A.~D ,~tl:-nt(ms. O p e n diamonds, [IZC]-Ieucine: open circles. [t~C]-
glutamicacid;closedcircles.[l~C]-glutamine. radioactivity into acid precipitable material (data not shown). Both amino acids gave significant incorporation into acid precipitable material under aminoacylation conditions, indicating that biologically active aminoacyl-tRNA synthetases are present in this cell-free preparation (Fig. 1). The difference in yield of aminoacyl tRNAs for the reactions performed with leucine versus glutamic acid can be explained by differences in the relative sizes of the two tRNA acceptor families present in our bulk preparation, it is thus possible to control endogenous proteases and nucleases in the extracts from this organism, and to prepare extracts that are capable of the initial step of protein biosynthesis, i.e. aminoacylation of tRNAs.
4.Z S. coelicolor is lacking glutamblyl-tRNA synthetase actirity With S. coelicolor being a Gram-positive bacterium, we were prompted to look for the misacylation pathway previously found in Bacillus species, mitochondria and chloroplasts [1,2,5], in this specialized organism. Aminoacylation experiments with bulk tRNA and a cell-free extract from S. coelicolor indicated that there was no detectable GInRS activity (Fig. 1). The reaction kinetics, as well as comparison to a control exper-
4.3. A tRNA specific amidotransferase actirity replaces GbzRS in S. coelicolor in all organisms and organelles investigated so far, the lack of GInRS is concomitant with the presence of a two-step pathway for the synthesis of glutamyi-tRNA¢;]". The fate of the tRNAbound glutamate in extracts from S. coelicoior was investigated. Incubation of [14C]-glutamate and tRNA under aminoacyiation conditions resulted in the conversion of about 50% of the input radioactivity into tRNA bound glutamine (Fig. 2), irrespective of the addition of [I-'C]glutaminc in the reaction. The conversion to glutaminyl-tRNA without added free glutamine is frequently found in crude extracts and indicates the incomplete removal of endogenous amino acids during extract preparation [4]. The tailing of the peaks for glutamate, and the slightly different R~ values for glutamate, are due to salt, nucleotides and the different amounts of [L'C]amino acids that are co-migrating in this area of the chromatogram under the conditions used. Incubation of [~4C]-glutamate under aminoacylation conditions, but without added tRNA, gave no conversion of glutamate to glutamine, indicating that the reaction is indeed a tRNA-dependent transamidation (data not shown). We can thus conclude that, in S. coelicolor, a tRNA-dependent amidotransferase is responsible for the formation of glutamyl-tRNAc'~" from the misacylated intermediate, glutamyl-tRNA6In. Whereas GInRS activity was not present in the extracts from S. coelicolor, it was shown that GluRS had the recognition characteristics of the
rate future investigations of possible regulatory roles of tRNAs in S. coelicolor [17].
Radi,,,r~ctwity detected 0 tO
50 cpm ,
,
ACKNOWLEDGEMENT -
E
Glu
-
Gin
0
The authors wish to thank Math(as Sprinzt in whose laboratory this work was performed. J.S.R was supported by an EMBO Short Term fellowship.
u
REFERENCES .
-Gin
+Gin
Fig. 2. Thin-layer chromatographic analysis of the tRNAbound amino acids. Samples were prepared and analysed as described in section 3.5. The sample lanes were scanned for [~4C]. Left lane, aminoacybtion assay without added [t:C]glutamine: right lane, aminoacylation assay with addition of I).33 mM [~'C]-glutamine. The marks to the right of the plate indicate the position of ',he chromatographic ,aandards; the bar at the top of the plate gives a scale for the amount of radioactivity detected in each lane. Chromatography was from bottom to top: only the lower 12 cm (starting from the point of sample application) of the chromatogram are shown.
homologous enzyme from Gram-positive bacteria and organelles, charging both tRNAGlu and tRNAGIn with glutamic acid. In addition to aminoaeylation, these extracts were active in the conversion of tRNA-bound glutamate to tRNAbound glutamine, indicating that the misacylation-transamidation pathway described for organelles and in other Gram-positive bacteria is a phenomenon that is also found in an organism that is as specialized in its biochemical and' morphological characteristics as S. coelicolor. The availability of a cell-free extract capable of tRNA-dependent metabolic reactions will facili-
[1] Wilcox. M. and Nirenber~, M. (1968)Proc. Natl. Acad, Sci. USA 61,229-236. [2] Martin, N.C., Rabinowitz, M. and Fukuhara, H, (1976) J. Mol. Biol. 101,285-296, 13] Gupta, R. (1985)J. Biol. Chem. 259, 94t51-9471. [4] Sch6n, A,, Hottinger. H. and $611, D. (1988) Biochimie 70, 391-394. [5] Sch6n, A., Kannaagara, C.G., Gough. S. and $811, D. (1988) Nature 331,187-190. [6] Lapp(rite, J., Duplain, L. and Proulx, M. (1986) J. Baeteriol. 165, 88-03. [7] Sch6n. A. and Si~il, D. (1988) FEI3S Lett. 228, 241-244. [8] Chert, M.-W., Jahn, D., Sch6n, A., O'lqeill, G.P. and $811, D. (1990) 1. Biol. Chem. 265, 4054-4057. [9] Thompson, CJ., Ward, J.M. and Hopwood, D.A. (19821 J. Bacteriol. 152, 668-677. [10] Okanishi, M., Ohta, T. and Umezawa, H. (1971)) J. Anlib(or. 23, 45-47. Jill Osmani, S.A,F,, Ma~ston, A.O., Selmes, J.P., Chapman, A.G. and Scrutlon, M.C. (1981) Eur, J. Biochem. 118. 271-278. [12] Von der Haar, F. (1973) Eur. J. Biochem. 34, 84-90. [13] Whitaker, R.J. and Granum, P.R. (1980) Anal. Biochem. 109, 156-159. [14] Roe, B,A. (1975) Nucleic Acids Res. 2, 21-42. [15] Von der Haar, F. (1974) Methods Enzymol. 34, 163-171. l!61 Von Arx, E. and Neher, R. (1963) ]. Chromatogr. t2, 329-34l, [17] Guthrie, E,P, and Chater, K.F. (19~}) J. Bacteriol, 172, 6189-6193.
Published by Elsevier
FEMSLE 04818
Misaminoacylation and tRNA-dependent transamidation in Streptomyces coelicolor A3(2) Astrid Sch6n ~ and J. Stefan Rokem b a Laboratorium fiir Biochemie, Unil~er.~itiit Bayreuth, Bayreuth, FRG and ~ Department o]'Applied Microbiology. h~stitute ~f Micmbioh~h~', Jertt~ah'm. israel Received 28 October 1991 Revision received 10January 1992 Accepted 13 January 1992
Key words: Amidotransferase; Mischarging; Glutamyl-tRNA synthetase: Protein synthesis; Streptomyces; tRNA
1. SUMMARY
Streptomyces coelicolor was found to be devoid of glutaminyl-tRNA synthetase, in this bacterium, tRNA ~" is aminoacylated by glutamyltRNA synthetase to yield glutamyl-tRNATM, followed by correction to glutaminyl-tRNAc;tn by a tRNA-dcpendent amidotransferase.
2. INTRODUCTION The formation of glutamyl-tRNAC;In (GIntRNAG~"), the activated amino acid precursor re-
Correspondence to: A, Schi~n. Laboratorium fiir Biochemie. Universit~t Bayreuth, P.O. Box 10 12 51, 8580 Bayreuth, FRG.
quired for insertion of glutamine into polypeptide chains, proceeds via a two-step mechanism in Gram-positive bacteria, Archaebacteria, mitochondria and chloroplasts [1-5]. These sources do not possess any glutaminyl-tRNA synthetase (GlnRS), but instead contain a naturally mischarging glutamyl-tRNA synthetase (GIuRS}. This enzyme misacylates tRNA Gin with glutamate to yield GIu-tRNA c;~", which is subsequently convetted to GIn-tRNAc'l" by a tRNA dependent amidotransferase [1,6-8]. The transamidation requires, similar to aminoacyI-tRNA formation, the presence of Mg 2~ and the consumption of ATP, and in addition glutamine or asparagine as an amide group donor. In this communication we describe the preparation of a cell free extract capable of tRNA dependent reactions, and we show that this mischarging pathway and the subsequent transamidation to yield GIn-tRNAt~n is also used by the Gram-positive bacterium, Strep-
tomyces coelicolor.
3. MATERIALS AND METHODS All biochemicals were of the highest purity available. [~4C]-amino acids were from Amersham Radiochemicals or New England Nuclear.
3.1. Bacterial stra#~ and growth conditions S. coelicolor A3(2) was obtained from J. Piret (Northeastern University, Boston, MA, USA). Solid medium for growth of the bacterium was R2YE [9]. Liquid cuItures were grown in AM medium [10] to the late logarithmic growth phase. 3.2. Cell extract After harvesting by centrifugation, the ceils were suspended in 2 volumes of synthetase buffer (SB; 50 mM Tris. HCI pH 7.5; 1 mM Na2EDTA; 10 mM MgCI2; 10 mM /3-mercaptoethanol; 0.2 m'M PMSI;) with the aid of a 'Potter' type tissue homogenizer with Teflon pestle. The cells were opened by N, cavitation [11], and ribosomes and cell debris were removed by centrifugation at 100000 ×g and 4°C for 1 h. Endogenous tRNAs were removed by adjusting the extract to 0.3 M NaCI and passing it over DE 52 (Whatman) equilibrated with SB containing 0.3 M NaCI [12]. The postribosomal supernatant was dialysed against 2 × 500 volumes of SB containing 0.2 mM PMSF and 44% glycerol (v/v), and stored at -20°C. The protein concentration in the cell extract was estimated spectrophotometrically according to [13]. 3.3. tRNA preparation Cells grown as above were suspended in buffer A (0.14 M NaAc, pH 4,5) and disrupted directly into saturated phenol. The aqueous phase was re-extracted with phenol and washed with chloroform/isoamylalcohol (24: 1). The combined aqueous phases were bound to a DEAE cellulose column (Whatman DE 52, 1.5 × 5 cm) [14] equilibrated with buffer A, the column was washed with buffer A containing 0.3 M NaCI, and the tRNA fraction was eluted with buffer A containing 1 M NaCI. Nucleic acids were precipitated by addition of 2 volumes of ethanol, collected by centrifugation, dried under vacuum and dissolved in HzO. Bulk tRNA concentration was estimated
spectrophotometrically, with 1.6 nmol/ml tRNA corresponding to an A z~0 of 1.
3.4. Amincacylation assays Aminoacylation assays [15] were performed in 100 mM Tris. HCI, pH 7.5; 10 mM MgCI2; 4 mM fl-mcrcaptoethanol; 5 mM ATP; 15 p.M [~4C]amino acid (leucine, 342 mCi/mmol; glutamic acid, 293 mCi/mmol; glutamine, 287 mCi/mmol), 15 ~M bulk tRNA, and 1.5 mg/ml cell extract. 10-~1 aliquots were spotted on filter paper disks that were washed in 10% TCA, 5% TCA and ethanol. Radioactivity was detected by liquid scintillation counting. 3.5. Amidotransferase assays The composition of the complete amidotransferase assay was identical to the aminoacylation and contained, in addition, 0.33 mM [~2C]glutamine. The reactions were stopped by adjusting them to pH 4.5 and 0.2 M NaAc, followed by phenol extraction and precipitation of the aqueous phases. The nucleic acids were deacylated by incubation in 50 mM NaHCO 3 (pH 9.1) at 50°C for 15 min, and the resulting hydrolysate was dried down and applied to a cellulose TLC plate (20 cm long) at 1.5 cm from the edge of the plate. The chromatogram was developed in isopropanol:HCOOH:H_,O (80:4:20; [16]) and scanned for the presence of radioactivity with a Berthoid Scanner !I (model LB 2723).
4. RESULTS AND DISCUSSION
4.1. Preparation of a cell free extract from S. coelicolor capable of aminoacylation A system was established that allowed the investigation of possible tRNA-dependent regulatory mechanisms. We have chosen tRNAaminoacylation as an assay system for RNAmetabolic reactions since it is simple to monitor. Reaction conditions were optimised with leucine and glutamic acid as [14C]-Iabelled amino acids and S. coelicolor tRNA a~ substrate. A control experiment, where exogenous bulk tRNA was omitted from the reaction mixture containing [14C]-Ieucine, gave no significant incorporation of
A
Leu ..,.,.
~s "6
E ..,=4
~2 ,~
~
Gtu
ml "
2
5
,
10
= Gin
20
iment lacking tRNA (see 4.1.), clearly demonstrate that even after a prolonged incubation time, there was no significant incorporation of [14C]-glutamine into acid-precipitable material. In contrast, aminoacylation with [~4C]-glutamie acid r~:~ultcd in an appreciable amount of this amino acid bound to tRNA. It is thus likely that S. coelicolor, like all other Gram-positive bacteria investigated so far, do not possess GlnRS, but instead make use of a naturally misacylating GluRS and a two-step pathway for the synthesis of GIn-tRNA (;~" via a misacylated Giu-tRNA G~" intermediate [1,4].
time (min) Fig. I. Time course of aminoacylalion ol S. codii'olor IRNA with a postribosomal supernatanl from S. cot'licc~h~r. Reactions were performed as described in MA'n~RI,Xl,S A.~D ,~tl:-nt(ms. O p e n diamonds, [IZC]-Ieucine: open circles. [t~C]-
glutamicacid;closedcircles.[l~C]-glutamine. radioactivity into acid precipitable material (data not shown). Both amino acids gave significant incorporation into acid precipitable material under aminoacylation conditions, indicating that biologically active aminoacyl-tRNA synthetases are present in this cell-free preparation (Fig. 1). The difference in yield of aminoacyl tRNAs for the reactions performed with leucine versus glutamic acid can be explained by differences in the relative sizes of the two tRNA acceptor families present in our bulk preparation, it is thus possible to control endogenous proteases and nucleases in the extracts from this organism, and to prepare extracts that are capable of the initial step of protein biosynthesis, i.e. aminoacylation of tRNAs.
4.Z S. coelicolor is lacking glutamblyl-tRNA synthetase actirity With S. coelicolor being a Gram-positive bacterium, we were prompted to look for the misacylation pathway previously found in Bacillus species, mitochondria and chloroplasts [1,2,5], in this specialized organism. Aminoacylation experiments with bulk tRNA and a cell-free extract from S. coelicolor indicated that there was no detectable GInRS activity (Fig. 1). The reaction kinetics, as well as comparison to a control exper-
4.3. A tRNA specific amidotransferase actirity replaces GbzRS in S. coelicolor in all organisms and organelles investigated so far, the lack of GInRS is concomitant with the presence of a two-step pathway for the synthesis of glutamyi-tRNA¢;]". The fate of the tRNAbound glutamate in extracts from S. coelicoior was investigated. Incubation of [14C]-glutamate and tRNA under aminoacyiation conditions resulted in the conversion of about 50% of the input radioactivity into tRNA bound glutamine (Fig. 2), irrespective of the addition of [I-'C]glutaminc in the reaction. The conversion to glutaminyl-tRNA without added free glutamine is frequently found in crude extracts and indicates the incomplete removal of endogenous amino acids during extract preparation [4]. The tailing of the peaks for glutamate, and the slightly different R~ values for glutamate, are due to salt, nucleotides and the different amounts of [L'C]amino acids that are co-migrating in this area of the chromatogram under the conditions used. Incubation of [~4C]-glutamate under aminoacylation conditions, but without added tRNA, gave no conversion of glutamate to glutamine, indicating that the reaction is indeed a tRNA-dependent transamidation (data not shown). We can thus conclude that, in S. coelicolor, a tRNA-dependent amidotransferase is responsible for the formation of glutamyl-tRNAc'~" from the misacylated intermediate, glutamyl-tRNA6In. Whereas GInRS activity was not present in the extracts from S. coelicolor, it was shown that GluRS had the recognition characteristics of the
rate future investigations of possible regulatory roles of tRNAs in S. coelicolor [17].
Radi,,,r~ctwity detected 0 tO
50 cpm ,
,
ACKNOWLEDGEMENT -
E
Glu
-
Gin
0
The authors wish to thank Math(as Sprinzt in whose laboratory this work was performed. J.S.R was supported by an EMBO Short Term fellowship.
u
REFERENCES .
-Gin
+Gin
Fig. 2. Thin-layer chromatographic analysis of the tRNAbound amino acids. Samples were prepared and analysed as described in section 3.5. The sample lanes were scanned for [~4C]. Left lane, aminoacybtion assay without added [t:C]glutamine: right lane, aminoacylation assay with addition of I).33 mM [~'C]-glutamine. The marks to the right of the plate indicate the position of ',he chromatographic ,aandards; the bar at the top of the plate gives a scale for the amount of radioactivity detected in each lane. Chromatography was from bottom to top: only the lower 12 cm (starting from the point of sample application) of the chromatogram are shown.
homologous enzyme from Gram-positive bacteria and organelles, charging both tRNAGlu and tRNAGIn with glutamic acid. In addition to aminoaeylation, these extracts were active in the conversion of tRNA-bound glutamate to tRNAbound glutamine, indicating that the misacylation-transamidation pathway described for organelles and in other Gram-positive bacteria is a phenomenon that is also found in an organism that is as specialized in its biochemical and' morphological characteristics as S. coelicolor. The availability of a cell-free extract capable of tRNA-dependent metabolic reactions will facili-
[1] Wilcox. M. and Nirenber~, M. (1968)Proc. Natl. Acad, Sci. USA 61,229-236. [2] Martin, N.C., Rabinowitz, M. and Fukuhara, H, (1976) J. Mol. Biol. 101,285-296, 13] Gupta, R. (1985)J. Biol. Chem. 259, 94t51-9471. [4] Sch6n, A,, Hottinger. H. and $611, D. (1988) Biochimie 70, 391-394. [5] Sch6n, A., Kannaagara, C.G., Gough. S. and $811, D. (1988) Nature 331,187-190. [6] Lapp(rite, J., Duplain, L. and Proulx, M. (1986) J. Baeteriol. 165, 88-03. [7] Sch6n. A. and Si~il, D. (1988) FEI3S Lett. 228, 241-244. [8] Chert, M.-W., Jahn, D., Sch6n, A., O'lqeill, G.P. and $811, D. (1990) 1. Biol. Chem. 265, 4054-4057. [9] Thompson, CJ., Ward, J.M. and Hopwood, D.A. (19821 J. Bacteriol. 152, 668-677. [10] Okanishi, M., Ohta, T. and Umezawa, H. (1971)) J. Anlib(or. 23, 45-47. Jill Osmani, S.A,F,, Ma~ston, A.O., Selmes, J.P., Chapman, A.G. and Scrutlon, M.C. (1981) Eur, J. Biochem. 118. 271-278. [12] Von der Haar, F. (1973) Eur. J. Biochem. 34, 84-90. [13] Whitaker, R.J. and Granum, P.R. (1980) Anal. Biochem. 109, 156-159. [14] Roe, B,A. (1975) Nucleic Acids Res. 2, 21-42. [15] Von der Haar, F. (1974) Methods Enzymol. 34, 163-171. l!61 Von Arx, E. and Neher, R. (1963) ]. Chromatogr. t2, 329-34l, [17] Guthrie, E,P, and Chater, K.F. (19~}) J. Bacteriol, 172, 6189-6193.