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Synthesis of Low Molecular Weight (5 S and 4 S) RNA in Isolated Spinach Chloroplasts
Biochem. Physiol. Pflanzen 169, S. 523-526 (1976)
Short Communication
Synthesis of Low Molecular \Veight (5 Sand 4 S) RNA in Isolated Spinach Chloroplasts E. SERFLING and K. ADLER Zentralinstitut fiir Genetik und Kulturpflanzenforschung der AdW, Gatersleben Key Term Index: chloroplast, RNA biosynthesis, low molecular weight RNA; Spina cia oleracea.
Summary In vitro chloroplasts synthesize under appropriate conditions besides high-molecular weight RNA low molecular weight 5 S RNA and 4 S RNA. It is concluded that these low molecular weight RNAs are coded for by the chloroplast DNA.
Chloroplasts like mitochondria contain a semi-autonomous protein-synthesizing system which in many respects resembles the protein-synthesizing machinery of prokaryotes (see P ARTHIER 1970; SMILLIE 1972, for reviews). The ribosomes of chloroplasts are, e. g., significantly smaller in size than cytoplasmic ribosomes and contain ribosomal RNA molecules of 23 S (1.1 X 106 D) and 16 S (0.56 x 106 D) (LOENING and INGLE 1967) typical for bacterial ribosomes (LOENING 1968), and no 5.8 S RNA component (PAYNE and DYER 1972). Recently it has been shown by nucleic acid hybridization that genes coding for chloroplast ribosomal 23 Sand 16 S RNA fractions reside in chloroplast DNA (TEWARI and WILDMAN 1968; INGLE et al. 1971; THOMAS and TEWARl1974). The 5 S RNA of chloroplasts can be distinguished from cytoplasmic 5 S RNA by gel electrophoresis (PAYNE and DYER 1971). As shown by sequence analysis, however. chloroplast 5 S RNA like cytoplasmic 5 S RNA comprises about 120 nucleotides (JORDAN and GALLING 1972), and apparently differs from the latter by its molecular conformation. Until now there is no experimental evidence whether chloroplast 5 S RNA is coded for by nuclear DNA or by chloroplast DNA. In this report, it will be shown that chloroplast 5 S RNA is apparently transcribed from chloroplast DNA since 5 S RNA is synthesized by chloroplasts in vitro. Spinach chloroplasts were isolated according to the method of HARTLEY and ELLIS (1973). During incubation for 30 min newly synthesized RNA was labelled by tritiated ATP and extracted as described in the legend to fig. 1. As reported by other authors (WOLLGIEHN and MUNSCHE 1972), the in vitro labelling of chloroplast RNA was significantly stimulated by use of 3H-ATP as radioactive precursor (about 10-20 fold in contrast to 3H-uridine labelling) and addition of the other triphosphates.
In Fig.la typical result of electrophoretic separation of in vitro synthesized spinach chloroplast RNA is shown. The distribution of radioactivity of 2.4% acrylamide gels reveals a rather heterogeneous profile with significant peaks in the size range of low 35*
524
E. SERF LING and K. ADLER
molecular weig ht RNA (arou nd 4 S) and of ribosomal RNAs, main ly of abou t 25 S (1.3 X 106 D). Since we detec ted low molecular weig ht RNA in several expe rime nts after 3H-uridine labelling as well it can be excluded that the label of low molecular weight RNA .is due to a nucle otidy l trans feras e activ ity. RNA molecule'! of 1.3 X 106 D are the main components of all spina ch plast id RNA prep arati ons labelled for 20-3 0 min in vitro (unp ublis hed results). From our findings and numerous repor ts in the litera ture (e. g. CARR ITT and EISEN S'I'AD T 1973; DETC HON and POSS INGH AM 1973; WOL LGIE HN and MUN SCHE 1972) it may be concluded that this fract ion repre sents the imm ediat e precursors of matu re 23 S rRNA. Besides these RNA fract ions significant large r RNA molecules were detec ted forming RNA peaks at about
235
~
1
165
~
2000
Z'
·S
] ~
1000
200
10
20
.30
40
50
60
70
80 slice-no.
Fig. 1. Electrophoretic spara tion of chloroplast RNA labelled in vitro. Spina ch leaves were washe d in sterile water and homo geniz ed for 10 sec in sterili zed 25 mM HEPES buffe r, pH 7.6, 0.35 M sucrose, 2 mM EDTA (HARTLEY and ELLIS 1973). The slurry was filtered throu gh two layers of germfree nylon , centri fuged (2.3 x 103 x g) for 1 min at 4°C and resusp ended in steril e 30 mM HEPES buffe r, pH 8.3, 6.6 mM MgCl 2 , 0.2 M KCl and 1 mg per ml Macaloid as RNase inhib itor; UTP, CTP, GTP (lO.um each) and 3H-A TP (15 CifmM , lO.uCifml; Radio chem ical Centre Amer sham) . The chloro plasts were incub ated in an illum inated therm ostat at 25°C for 30 min. After incub ation, chloro plasts were centri fuged , dissolved in lysis buffer (0.01 M Tris, pH 7.6; 0.1 M NaCI; 0.01 M MgCl2 ; 0.2 % SDS) and centri fuged again . The super natan t was mixed with an equal vol. of a freshl y prepa red 1: 1 mixtu re of pheno lfm-c resole f8-hy droxy quino line (HAST INGS and KIRBY 1966) and chloro formf isoam ylalco hol (24: 1) and depro teiniz ed three times by stirrin g at 4 °C for 20 min each. The RNA was preci pitate d overn ight by 3 vol. of ethan ol, purifi ed on a column of Sepha dex G-50 and, before electr ophor esis passe d throu gh a column of oligo-(dT)-cellulo se (AVIV and LEDER 1972). Electr ophor esis of RNA not bound to the cellulose was carrie d out on a 2.4% acryla mide gel prepa red accor ding to BISHO P et al. (1967) for 3 h at 4°C. After the run, the gels were staine d and fixed in a soluti on of methy lene blue and sodiu m acetat e-ace tic acid (1: 1) at room tempe rature and destai ned in distill ed water . The gels were frozen over dry ice in hexan , cut into I-mm pieces, and the positi on of staine d bands was noted . Slices were dissolved in 0.2 ml of 30% H 2 0 2 at 55°C in scinti llatio n vials. After addit ion of 7 ml Bray' s soluti on (BRAY 1960) radioa ctivity was measu red in a P AOKARD Trica rb liquid scinti llatio n spectr omete r.
Synthesis of Low Molecular Weight RNA in Chloroplasts
525
2.7 X 106 D as shown in Fig. 1 and reported by HARTLEY and ELLIS (1973) or, in some cases, at intermediate positions. The low molecular weight RNA found in preparations of chloroplast RNA labelled in vitro can be further fractionated on 7 -10 % acrylamide gels. A typical radioactivity pattern obtained after fractionation of chloroplast RNA on a 10 % urea-acrylamide gel is given in Fig. 2. In spite of a rather high background labelling (which was observed in the majority of our RNA preparations labelled in vitro) both in position of 4 S RNA and 5 S RNA a clear-cut radioactive peak is detected. Resistance of 4 Sand 5 S RNA fractions against heating in 8 M urea demonstrates that both molecule classes contain intact molecules and no arteficial aggregates which may be formed by hydrogen bonds between smaller molecules (REIJNDERS et al. 1973). Several lines of evidence suggest that in our chloroplast preparation the synthesis of 4 S RNA and 5 S RNA is neither due to bacterial contamination nor to residual nculear RNA synthesis. In all experiments the leaves were carefuly washed with sterile water; buffers and incubation media were filtered through Schott G-5 bacteria filters and all glassware was sterilized by autoclaving. So the ratio of chloroplasts: bacteria in the incubation medium was drastically reduced and was estimated to be 600: 1. Under the experimental conditions used (short time lysis of chloroplasts in the presence of low detergent concentrations, see WOLLGIEHN et al. 1974), only RNA of chloroplasts should be extracted, whereas both the stimulation of RNA synthesis by triphosphates, the
Fig. 2. Electrophoretic separation of low molecular weight chloroplast RNA labelled in vitro. Chloroplast RNA, labelled and isolated as described in fig. 1, was heated for 2 min at 60°C in electrophoresis buffer containing 8 M urea and fractionated on a 10 % urea-acrylamide gel (REIJNDERS et al. 1973) for 2.5 h.
526
E. SERFLING and K. ADLER, Synthesis of Low Molecular Weight RNA in Chloroplasts
labelling kinetics and the electrophoretic mobility of RNA molecules (including 5 S RNA) support our conclusion of synthesis of chloroplast-specific low molecular weight RNA molecules. References AVIV, H., and LEDER, P., Purification of biologically active globin messenger RNA by chromatograph on oligothymidylic acid cellulose. Proc. Nat. Acad. Sci. 69, 1408-1412 (1972). BISHOP, D. H. L., CLAYBRCOK, J. L., and SPIEGELMAN', S., Electrophoretic separation of viral nucleic acids on polyacrylamide gels. J. Mol. BioI. 26, 373-387 (1967). BRAY, G. A., A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Anal. Biochem. 1, 279-285 (1960). CARRITT, B., and EISENSTADT, J. M., RNA synthesis in isolated chloroplasts: Characterization of the newly synthesized RNA. FEBS Letters 36,116-120 (1973). DETCHON', P., and POSSINGHAM, J. P., Chloroplast ribosomal ribonucleic acid synthesis in cultured spinach leaf tissue. Biochem. J. 136, 829-836 (1973). HARTLEY, M. R., and ELLIS, R. J., Ribonucleic acid synthesis in chloroplasts. Biochem. J. 134, 249-262 (1973). HASTIN'GS, J. R. B., and KIRBY, K. S., The nucleic acids of Drosophila melanogaster. Biochem. J. 100, 532-539 (1966). INGLE, J., WELLS, R., POSSINGHAM, J. V., and LEAVER, C. J., The origins of chloroplast ribosomalRNA. Symp. Austr. Acad. Sci. and U.S. Nat!. Acad. Sci. 1971, 393-401. JORDAN', B. R., and GALLIN'G, G., Zur Basensequenz cytoplasmatischer und chloroplastidarer 5 S RNA. Ber. Dtsch. Bot. Ges. 85, 383-389 (1972). LOEN'ING, U., Molecular weights of ribosomal RNA in relation to evolution. J. Mol. BioI. 38, 355-365 (1968). LOENING, U. E., and INGLE, J., Diversity of RNA components in green plant tissues. Nature 2Ui, 363-367 (1967). P ARTHIER, B., Existenz und Realisierung extrachromosomaler genetischer Information in Plastiden und Mitochondrien. BioI. Rdsch. 8, 289-306 (1970). PAYN'E, P. I., and DYER, T. A., Plant 5.8 S RNA is a component of 80 S but not 70 S ribosomes. Nature 231), 145-147 (1972). - - Characterization of cytoplasmic and chloroplast 5 S ribosomal ribonucleic acid from broadbean leaves. Biochem. J. 124, 83-89 (1971). REIJNDERS, L., SLOOF, P., SIVAL, J., and BORST, P., Gel Electrophoresis of RNA under denaturing conditions. Biochim. Biophys. Acta 324, 320-333 (1973). SMILLIE, R. M., Synthesizing capability of the photosynthetic apparatus. Proteins. Methods in Enzymology 24 (B), 381- 394 (1972). THOMAS, J. R., and TEWARI, K. K., Ribosomal RNA genes in the chloroplast DNA of pea leaves. Biochim. Biophys. Acta 261,73-83 (1974). WOLLGIEHN, R., and MUNSCHE, D., RNS-Synthese in isolierten Chloroplasten von Nicotiana rustica. Biochem. Physiol. Pflanzen 163, 137 -155 (1972). - und MIKULOWITSCH, T. P., 32P-Inkorporation in die Chloroplasten-RNS von Tabakblattern. Ausschlu6 der Beteiligung von Bakterien-Verunreinigungen. Biochem. Physiol. Pflanzen 160, 37-48 (1974). Received October 27, Revision December 22,1975. Authors' address: Dr. E. SERFLING and Dr. K. ADLER, Zentralinstitut fiir Genetik und Kulturpflanzenforschung der AdW, 4325 Gatersleben.
Short Communication
Synthesis of Low Molecular \Veight (5 Sand 4 S) RNA in Isolated Spinach Chloroplasts E. SERFLING and K. ADLER Zentralinstitut fiir Genetik und Kulturpflanzenforschung der AdW, Gatersleben Key Term Index: chloroplast, RNA biosynthesis, low molecular weight RNA; Spina cia oleracea.
Summary In vitro chloroplasts synthesize under appropriate conditions besides high-molecular weight RNA low molecular weight 5 S RNA and 4 S RNA. It is concluded that these low molecular weight RNAs are coded for by the chloroplast DNA.
Chloroplasts like mitochondria contain a semi-autonomous protein-synthesizing system which in many respects resembles the protein-synthesizing machinery of prokaryotes (see P ARTHIER 1970; SMILLIE 1972, for reviews). The ribosomes of chloroplasts are, e. g., significantly smaller in size than cytoplasmic ribosomes and contain ribosomal RNA molecules of 23 S (1.1 X 106 D) and 16 S (0.56 x 106 D) (LOENING and INGLE 1967) typical for bacterial ribosomes (LOENING 1968), and no 5.8 S RNA component (PAYNE and DYER 1972). Recently it has been shown by nucleic acid hybridization that genes coding for chloroplast ribosomal 23 Sand 16 S RNA fractions reside in chloroplast DNA (TEWARI and WILDMAN 1968; INGLE et al. 1971; THOMAS and TEWARl1974). The 5 S RNA of chloroplasts can be distinguished from cytoplasmic 5 S RNA by gel electrophoresis (PAYNE and DYER 1971). As shown by sequence analysis, however. chloroplast 5 S RNA like cytoplasmic 5 S RNA comprises about 120 nucleotides (JORDAN and GALLING 1972), and apparently differs from the latter by its molecular conformation. Until now there is no experimental evidence whether chloroplast 5 S RNA is coded for by nuclear DNA or by chloroplast DNA. In this report, it will be shown that chloroplast 5 S RNA is apparently transcribed from chloroplast DNA since 5 S RNA is synthesized by chloroplasts in vitro. Spinach chloroplasts were isolated according to the method of HARTLEY and ELLIS (1973). During incubation for 30 min newly synthesized RNA was labelled by tritiated ATP and extracted as described in the legend to fig. 1. As reported by other authors (WOLLGIEHN and MUNSCHE 1972), the in vitro labelling of chloroplast RNA was significantly stimulated by use of 3H-ATP as radioactive precursor (about 10-20 fold in contrast to 3H-uridine labelling) and addition of the other triphosphates.
In Fig.la typical result of electrophoretic separation of in vitro synthesized spinach chloroplast RNA is shown. The distribution of radioactivity of 2.4% acrylamide gels reveals a rather heterogeneous profile with significant peaks in the size range of low 35*
524
E. SERF LING and K. ADLER
molecular weig ht RNA (arou nd 4 S) and of ribosomal RNAs, main ly of abou t 25 S (1.3 X 106 D). Since we detec ted low molecular weig ht RNA in several expe rime nts after 3H-uridine labelling as well it can be excluded that the label of low molecular weight RNA .is due to a nucle otidy l trans feras e activ ity. RNA molecule'! of 1.3 X 106 D are the main components of all spina ch plast id RNA prep arati ons labelled for 20-3 0 min in vitro (unp ublis hed results). From our findings and numerous repor ts in the litera ture (e. g. CARR ITT and EISEN S'I'AD T 1973; DETC HON and POSS INGH AM 1973; WOL LGIE HN and MUN SCHE 1972) it may be concluded that this fract ion repre sents the imm ediat e precursors of matu re 23 S rRNA. Besides these RNA fract ions significant large r RNA molecules were detec ted forming RNA peaks at about
235
~
1
165
~
2000
Z'
·S
] ~
1000
200
10
20
.30
40
50
60
70
80 slice-no.
Fig. 1. Electrophoretic spara tion of chloroplast RNA labelled in vitro. Spina ch leaves were washe d in sterile water and homo geniz ed for 10 sec in sterili zed 25 mM HEPES buffe r, pH 7.6, 0.35 M sucrose, 2 mM EDTA (HARTLEY and ELLIS 1973). The slurry was filtered throu gh two layers of germfree nylon , centri fuged (2.3 x 103 x g) for 1 min at 4°C and resusp ended in steril e 30 mM HEPES buffe r, pH 8.3, 6.6 mM MgCl 2 , 0.2 M KCl and 1 mg per ml Macaloid as RNase inhib itor; UTP, CTP, GTP (lO.um each) and 3H-A TP (15 CifmM , lO.uCifml; Radio chem ical Centre Amer sham) . The chloro plasts were incub ated in an illum inated therm ostat at 25°C for 30 min. After incub ation, chloro plasts were centri fuged , dissolved in lysis buffer (0.01 M Tris, pH 7.6; 0.1 M NaCI; 0.01 M MgCl2 ; 0.2 % SDS) and centri fuged again . The super natan t was mixed with an equal vol. of a freshl y prepa red 1: 1 mixtu re of pheno lfm-c resole f8-hy droxy quino line (HAST INGS and KIRBY 1966) and chloro formf isoam ylalco hol (24: 1) and depro teiniz ed three times by stirrin g at 4 °C for 20 min each. The RNA was preci pitate d overn ight by 3 vol. of ethan ol, purifi ed on a column of Sepha dex G-50 and, before electr ophor esis passe d throu gh a column of oligo-(dT)-cellulo se (AVIV and LEDER 1972). Electr ophor esis of RNA not bound to the cellulose was carrie d out on a 2.4% acryla mide gel prepa red accor ding to BISHO P et al. (1967) for 3 h at 4°C. After the run, the gels were staine d and fixed in a soluti on of methy lene blue and sodiu m acetat e-ace tic acid (1: 1) at room tempe rature and destai ned in distill ed water . The gels were frozen over dry ice in hexan , cut into I-mm pieces, and the positi on of staine d bands was noted . Slices were dissolved in 0.2 ml of 30% H 2 0 2 at 55°C in scinti llatio n vials. After addit ion of 7 ml Bray' s soluti on (BRAY 1960) radioa ctivity was measu red in a P AOKARD Trica rb liquid scinti llatio n spectr omete r.
Synthesis of Low Molecular Weight RNA in Chloroplasts
525
2.7 X 106 D as shown in Fig. 1 and reported by HARTLEY and ELLIS (1973) or, in some cases, at intermediate positions. The low molecular weight RNA found in preparations of chloroplast RNA labelled in vitro can be further fractionated on 7 -10 % acrylamide gels. A typical radioactivity pattern obtained after fractionation of chloroplast RNA on a 10 % urea-acrylamide gel is given in Fig. 2. In spite of a rather high background labelling (which was observed in the majority of our RNA preparations labelled in vitro) both in position of 4 S RNA and 5 S RNA a clear-cut radioactive peak is detected. Resistance of 4 Sand 5 S RNA fractions against heating in 8 M urea demonstrates that both molecule classes contain intact molecules and no arteficial aggregates which may be formed by hydrogen bonds between smaller molecules (REIJNDERS et al. 1973). Several lines of evidence suggest that in our chloroplast preparation the synthesis of 4 S RNA and 5 S RNA is neither due to bacterial contamination nor to residual nculear RNA synthesis. In all experiments the leaves were carefuly washed with sterile water; buffers and incubation media were filtered through Schott G-5 bacteria filters and all glassware was sterilized by autoclaving. So the ratio of chloroplasts: bacteria in the incubation medium was drastically reduced and was estimated to be 600: 1. Under the experimental conditions used (short time lysis of chloroplasts in the presence of low detergent concentrations, see WOLLGIEHN et al. 1974), only RNA of chloroplasts should be extracted, whereas both the stimulation of RNA synthesis by triphosphates, the
Fig. 2. Electrophoretic separation of low molecular weight chloroplast RNA labelled in vitro. Chloroplast RNA, labelled and isolated as described in fig. 1, was heated for 2 min at 60°C in electrophoresis buffer containing 8 M urea and fractionated on a 10 % urea-acrylamide gel (REIJNDERS et al. 1973) for 2.5 h.
526
E. SERFLING and K. ADLER, Synthesis of Low Molecular Weight RNA in Chloroplasts
labelling kinetics and the electrophoretic mobility of RNA molecules (including 5 S RNA) support our conclusion of synthesis of chloroplast-specific low molecular weight RNA molecules. References AVIV, H., and LEDER, P., Purification of biologically active globin messenger RNA by chromatograph on oligothymidylic acid cellulose. Proc. Nat. Acad. Sci. 69, 1408-1412 (1972). BISHOP, D. H. L., CLAYBRCOK, J. L., and SPIEGELMAN', S., Electrophoretic separation of viral nucleic acids on polyacrylamide gels. J. Mol. BioI. 26, 373-387 (1967). BRAY, G. A., A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Anal. Biochem. 1, 279-285 (1960). CARRITT, B., and EISENSTADT, J. M., RNA synthesis in isolated chloroplasts: Characterization of the newly synthesized RNA. FEBS Letters 36,116-120 (1973). DETCHON', P., and POSSINGHAM, J. P., Chloroplast ribosomal ribonucleic acid synthesis in cultured spinach leaf tissue. Biochem. J. 136, 829-836 (1973). HARTLEY, M. R., and ELLIS, R. J., Ribonucleic acid synthesis in chloroplasts. Biochem. J. 134, 249-262 (1973). HASTIN'GS, J. R. B., and KIRBY, K. S., The nucleic acids of Drosophila melanogaster. Biochem. J. 100, 532-539 (1966). INGLE, J., WELLS, R., POSSINGHAM, J. V., and LEAVER, C. J., The origins of chloroplast ribosomalRNA. Symp. Austr. Acad. Sci. and U.S. Nat!. Acad. Sci. 1971, 393-401. JORDAN', B. R., and GALLIN'G, G., Zur Basensequenz cytoplasmatischer und chloroplastidarer 5 S RNA. Ber. Dtsch. Bot. Ges. 85, 383-389 (1972). LOEN'ING, U., Molecular weights of ribosomal RNA in relation to evolution. J. Mol. BioI. 38, 355-365 (1968). LOENING, U. E., and INGLE, J., Diversity of RNA components in green plant tissues. Nature 2Ui, 363-367 (1967). P ARTHIER, B., Existenz und Realisierung extrachromosomaler genetischer Information in Plastiden und Mitochondrien. BioI. Rdsch. 8, 289-306 (1970). PAYN'E, P. I., and DYER, T. A., Plant 5.8 S RNA is a component of 80 S but not 70 S ribosomes. Nature 231), 145-147 (1972). - - Characterization of cytoplasmic and chloroplast 5 S ribosomal ribonucleic acid from broadbean leaves. Biochem. J. 124, 83-89 (1971). REIJNDERS, L., SLOOF, P., SIVAL, J., and BORST, P., Gel Electrophoresis of RNA under denaturing conditions. Biochim. Biophys. Acta 324, 320-333 (1973). SMILLIE, R. M., Synthesizing capability of the photosynthetic apparatus. Proteins. Methods in Enzymology 24 (B), 381- 394 (1972). THOMAS, J. R., and TEWARI, K. K., Ribosomal RNA genes in the chloroplast DNA of pea leaves. Biochim. Biophys. Acta 261,73-83 (1974). WOLLGIEHN, R., and MUNSCHE, D., RNS-Synthese in isolierten Chloroplasten von Nicotiana rustica. Biochem. Physiol. Pflanzen 163, 137 -155 (1972). - und MIKULOWITSCH, T. P., 32P-Inkorporation in die Chloroplasten-RNS von Tabakblattern. Ausschlu6 der Beteiligung von Bakterien-Verunreinigungen. Biochem. Physiol. Pflanzen 160, 37-48 (1974). Received October 27, Revision December 22,1975. Authors' address: Dr. E. SERFLING and Dr. K. ADLER, Zentralinstitut fiir Genetik und Kulturpflanzenforschung der AdW, 4325 Gatersleben.