ON THE BIOSYNTHESIS
OF BACTERIAL
RIBOSOMAL
RNA
T . J . STOOF AND R . J . PLANTA
Biochemisch Laboratorium, Vrije Universiteit, Amsterdam, The Netherlands
(Received 20 November, 1973) ABSTRACT. By comparison of the fingerprints of 5 S and 23 S ribosomal RNAs from Bacillus licheniformis with that of the precursor of 23 S ribosomal RNA, it can be shown that 5 S RNA is not a part of the precursor of 23 S ribosomal RNA. I. I N T R O D U C T I O N The genetic organization of the cistrons for bacterial ribosomal RNA species (23S, 16S and 5 S rRNA) has been studied in detail with Escherichia coli, Bacillus subtilis and B. megaterium [1-12]. These studies have shown that: (a) the number of gene copies for each RNA species is the same [1, 2], and (b) the 23 S, 16 S and 5 S rRNA genes are tightly linked and arranged as a block in the order 16 S-23 S-5 S [3-8]. Substantial evidence [5-8] has been obtained that each block of the linked three cistrons can be considered as a single 'transcriptional unit', in which transcription is initiated at the 16 S end and continues through the 23 S and 5 S RNA cistrons. Since no common RNA precursor of the three rRNA species has ever been found in bacteria, this model implies that the 16 S moiety is liberated from the growing RNA chain before transcription of the unit is complete. In fact, newly formed 16S and 23 S rRNAs are found first in precursor forms: the precursor form of 16S rRNA (designated as p l6S) is approximately 10~o larger than mature 16 S rRNA, and the precursor form of 23 S rRNA (p 23 S) is assumed to be about 200 nucleotides larger than mature 23 S rRNA [9-11 ]. Furthermore, it was inferred from several lines of indirect evidence that 5 SRNA is also transcribed as part of a rather large precursor molecule [4, 5, 7, 8, 12]. Taking these data together, it was tempting to propose that 5 S RNA, which in E. coli is only 120 nucleotides long [13], is part of p23S RNA, as was done by a number of authors [5, 7, 9, 11]. In this concept, the first transcription products from the bacterial ribosomal transcriptional unit are p l 6 S RNA and p23S RNA, after which 5S RNA is generated during the conversion of p23S R NA into 23S rRNA. We tested this hypothesis for B. licheniformis by comparing the fingerprints of 5 S, 23 S and p23S RNA with each other, and, as will be described in the present report, this led us to the conclusion that the 5 S RNA sequence is not present in the precursor of 23 S rRNA. II. MATERIALS A N D M E T H O D S Bacillus licheniformis (strain $244) was used in the present investigations. The bacteria were routinely grown at 37 ~ under vigorous aeration in a synthetic medium as previously described [14], or in a low-phosphate medium (see below). 243 Molecular Biology Reports 1 (1973) 243-249. All Rights Reserved Copyright 9 1973 by D. Reidel Publishing Company, Dordrecht-Holland
32p-labelled 23 S and 5 S rRNAs were obtained by adding 32p-orthophosphate to a final concentration of 0.05 mC ml- ~ to an early-exponential culture of B. licheniformis in a low-phosphatemedium (containing per litre: 6.05 g Tris-HC1, 50 mg MgSO4 97H20, 5 mg FeSO4, 2 g NH4C1, 1 g L-glutamic acid, 2 g KCI and 4.5 g standard bouillon, pH 7.3), continuing growth for 3 generations, collecting of cells by centrifugation at 3000 x g for l0 min, converting the cells to spheroplasts by treatment with lysozyme as described previously [14], and extracting RNA from the spheroplasts with phenol-SDS in essentially the same way as described by Ret 61 and Planta I15], except that the extraction was performed at 0 ~ The individual RNA species were separated by sucrose-gradient centrifugation in a Spinco SW 25-1 rotor at 22500 rpm for 16 hr [15]. The 23S rRNA was isolated from the appropriate fractions by ethanol-precipitation overnight at - 2 0 ~ and further purified by recentrifugation. As judged by electrophoretic analysis on 3~ polyacrylamide gels [l 6], the 23 S rRNA preparation thus obtained was practically homogeneous (contamination with 16 S rRNA was less than 2~). The 5 S RNA was purified by precipitation of the slowly sedimenting material from the appropriate gradient fractions with 3 vol of ethanol at - 2 0 ~ (overnight), and subsequent electrophoresis of the RNA sample on 10~ polyacrylamide gels [17]. The gels were cut in 1 mm slices, which were counted directly in a liquid scintillation counter. The slices containing the aEp-labelled 5 S RNA were pooled and eluted by shaking in a minimal volume of a solution of 1 M NaC1, 0.01 M Tris-HCl, pH 7.0 at 4 ~ overnight. p23 S RNA, pulse-labelled with 32p-orthophosphate, was prepared by adding to a culture in the mid-logarithmic growth phase on a low-phosphate medium (see above) chloramphenicol (final concentration 300 Ixgml- 1), and 3 min later 32p_orthophosphate (final concentration 0.5 mC ml- 1). The cells were harvested, converted to spheroplasts, and the RNA was extracted as described above, p23 S RNA was purified by repeated sucrose gradient centrifugation to a nearly homogeneous state, as judged by electrophoretic analysis on 3~ polyacrylamide gels [16]. p23S RNA, pulse-labelled with 3H-uridine was obtained in a similar manner, except that the culture was grown on a normal synthetic medium [14]. The fingerprints were made as follows: aliquots of 20 lag RNA in 0.005 ml 0.01 M Tris-HCl (pH 8.0) were digested with Tl-ribonuclease (enzyme-substrate ratio of l: 10) and alkaline phosphatase (enzyme-substrate ratio of 1:5) at 37 ~ for 30 min. The digestion products were separated in two dimensions by electrophoresis according to Sanger et al. [l 8]. The fingerprints were visualized by radioautography (medical X-ray film, type R, 3M Italia SPA). In one case (see Section III), fingerprinting was applied to a mixture of 32p., 14C_ and 3H-labelled RNAs. In this particular case, all a2p-containing spots (detected by radioautography) were cut out and counted for 3H and 14C after a nearly complete decay of the 32p-radioactivity. To this end, each spot was incubated with 0.5 ml Nuclear Chicago Solubilizer (NCS) in a counting vial at 37 ~ overnight. After addition of l0 ml toluene-based scintillation fluid, the vials were stored at 4 ~ until the 32p-activity had disappeared, and then counted in a liquid scintillation counter. III. RESULTS AND DISCUSSION Fig. 1 shows a typical electrophoretic separation pattern of B. licheniformis RNA, pulse-labelled with (~4C-methyl)-methionine, in the presence of chloramphenicol. It is well-known that inhibition of cellular protein synthesis (by chloramphenicol) results in an accumulation of the precursor forms of bacterial rRNA [19]. The pulse-labelled RNA appear to consist of well-defined RNA species with a clearly lower electrophoretic mobility than the corresponding mature rRNA components. That these rapidly-labelled RNA species do represent precursor forms of the rRNA components, may be inferred from the fact that they are methylated, and from the observation 244
20
10
23S
15
"'10 v
I
e9 I
O x c-
5
5
s
"T
/
/
I i f I I J J I I I I I I I J I I I I I
16S
5 1 v
I
ff O •
%
t'e9
,4.a
E U ,r E
E
c~ "o
"13 I
I
I
2O
4O
6O
FRACTION NUMBER Fig. 1. Polyacrylamide gel electrophoresis of pulse-labelled R N A of B. licheniformis. Exponentially growing cells in a n o r m a l synthetic m e d i u m [14] were labelled for three generations with 3H-uridine. Subsequently, chloramphenicol was added, 3 m i n later followed by pulse-labelling with (14C-methyl) methionine in the same way as described for saP-labelling in Section II. The cells were converted into spheroplasts, and the R N A was extracted as described in Section II. The R N A mixture was subjected to electrophoresis on 3 700 polyacrylamide gels, to which glycerol (7 % v/v) was added in order to facilitate slicing of the gels. The gels were sliced into 1 mm-slices with a Joyce-Loebl gel slicer, and subsequently monitored for all- and 14C-radioactivity after incubation overnight at 37 ~ in 10 ml toluene-based scintillation fluid containing the usual scintillator and 3 % N C S (Nuclear Chicago Solubilizer).
that they are processed to the mature rRNA components after removal of the antibiotic from the culture (results not shown). Similar separation patterns as in Fig. 1 were obtained after pulselabelling of the RNA with 3H-uridine or 32p-orthophosphate. In order to investigate the possible relationship between p23 S RNA and 5 S RNA, we decided to make fingerprints of both RNA species, and to look whether or not the fingerprint of p23S RNA contains spots characteristic of 5 S RNA. For the isolation of relatively pure p23 S RNA with sufficient high specific activity we preferred sucrose gradient centrifugation above gel electrophoresis. To our experience, elution of high-molecular-weight RNA from gel slices of low polyacrylamide concentrations produces RNA preparations highly contaminated with acrylamide. The presence of this contaminant in the RNA preparation results in fingerprints with a very bad resolution, particularly in the region of the larger oligonucleotides. Therefore, we subjected 32p-pulse-labelled RNA to sucrose gradient centrifugation. The radioactive fractions, sedimenting faster than 23 S rRNA were pooled, and this material was recentrifuged in the same way. 245
30
S
~0 20
D
X
d 10
I
I
20 40 FRACTION NUMBER
I
60
Fig. 2. Gel electrophoretic analysis of partially purified a~P-labelled p 23 S RNA. Labelling, extraction and purification of this precursor R N A are described in Section II, the conditions of electrophoresis are presented under Fig. 1.
Fig. 3. Two-dimensional separation patterns (upper parts) of a digest of a2p-labelled 5 S r R N A and 23 S rRNA, respectively. The R N A components, isolated as described in Section II, were digested with both Tl-ribonuclease and alkaline phosphatase. The removal of the 3'-terminal phosphate group enhances the mobility of the oligonucleotides in the second dimension which is important in particular for the larger fragments. In this group of fragments an unique spot from 5S R N A can be observed (indicated by an arrow). Faint spots are also visible in the fingerprint of 23 S rRNA, which are most probably due to an incomplete action of Tl-ribonuclease resulting in oligonucleotides with a terminal cyclic phosphate. For experimental details, see Section II.
246
Fig. 2 shows the electrophoretic analysis of an aliquot of the p23S R N A preparation thus obtained. It is obvious that the p23 S R N A preparation is still contaminated with a small amount of 23 S rRNA. However, this contamination does not really interfere with our approach, since we are looking for the presence of unique 5 S R N A spots, that is to say which are not present in a fingerprint of 23 S rRNA. Fig. 3 demonstrates that the rather simple fingerprint of 5 S R N A does indeed possess a spot (marked with an arrow), not coinciding with any spot of the 23 S rRNA-fingerprint (for the sake
Fig. 4. Two-dimensional separation patterns of a combined digest of 8~P-labelled 23 S plus 5 S rRNA, and p 23 S RNA, respectively. For further details see Section II and the legend to Fig. 3. 247
of clarity only the upper halves of the fingerprints are shown, i.e. the region where the larger oligonucleotides are located, and where differences might be expected to occur). However, for our purpose it is absolutely necessary that this oligonucleotide is an internal fragment, and does not correspond to a sequence containing the 5'- or 3'-end group. This argument is the more pertinent, since cleavage of a hypothetical common precursor of 5 S and 23 S rRNA seems not to be the final step in the 5S RNA maturation process [20, 21]. To settle this point, 32p-labelled 5S RNA was digested with Tl-ribonuclease alone to avoid the removal of a possible terminal phosphate group, the digest was fractionated according to Sanger et al. [18], and the spot corresponding to the oligonucleotide in question was located by radioautography. The oligonucleotide was eluted from the paper with 30% triethylamine carbonate (pH 10.0). After desalting of the sample by electrophoresis, the material was hydrolyzed with 1 N KOH for 16 hr at 37~ and the hydrolysis products were analyzed by electrophoresis on Whatman 3 MM at pH 3.5. By this type of analysis it could be established (as will be published elsewhere) that the oligonucleotide contains a 5'-OHand a -G-3'-P-end, and therefore represents an internal fragment of 5 S RNA with the bruto base
composition (C6U3G). In Fig. 4 the combined fingerprint of 32p-labelled 23 S plus 5 S rRNA can be compared with the fingerprint obtained from 32p-labelled p23S RNA. As can be seen, the unique 5S RNA spot, which is marked with an arrow in the combined fingerprint of 23 S plus 5 S rRNA, is absent in the fingerprint of p23S. Therefore, we conclude that p23S RNA does not contain the 5S RNA sequence. This conclusion was supported by the result of the following experiment. We isolated p23S RNA, pulse-labelled with 3H-uridine, and mixed with a fixed amount of 23S rRNA, uniformly labelled with 14C-uracil, and a small amount of 5 S RNA, uniformly labelled with 32p-orthophosphate. This mixture was digested and fingerprinted as described in the Methods section. The 32p-labelled oligonucleotides, derived from 5S RNA, were located by radioautography. All those spots were cut out, and after the decay of the 32p-radioactivity were assayed for an- and 14C-radioactivity as described in Section II. Although in the great majority of the spots both 3H- and 14C-counts were found, both activities were nearly completely absent in the unique 5S RNA-spot described above. This confirms the idea that this oligonucleotide, derived from 5 S RNA, is neither present in 23 S rRNA, nor in p 23 S RNA, in agreement with the conclusion drawn above. ACKNOWLEDGEMENTS The present investigations have been partly sponsored by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for the Advancement of Pure Research (ZWO). The authors highly appreciate the excellent technical support given by Miss V. C. H. F. de Regt. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 248
Smith, I., Dubnau, D., Morell, P., and Marmur, J., J. Mol. Biol. 33, 123 (1968). Pace, B. and Pace, N. R., J. Bacteriol. 105, 142 (1971). Colli, W. and Oishi, M., Proc. Nat. Acad. Sci. U.S. 64, 642 (1969). Bleyman, M., Kondo, M., Hecht, N., and Woese, C. R., J. Bacteriol. 99, 535 (1969). Pato, M. L. and von Meyenburg, K., ColdSpring Harbor Symp. Quant. Biol. 35, 497 (1970). Colli, W., Smith, I., and Oishi, M., J. Mol. Biol. 56, 117 (1971). Doolittle, W. F. and Pace, N. R., Proc. Nat. Acad. Sci. U.S. 68, 1786 (1971). Dennis, P. P. and Bremer, H., J. Mol. Biol. 75, 145 (1973). Hecht, N. B., Bleyman, M., and Woese, C. R., Proc. Nat. Acad. Sci. U.S. 59, 1278 (1968).
10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
Adesnik, M. and Levinthal, C., J. Mol. Biol. 46, 281 (1969). Pace, B., Peterson, R. L., and Pace, N. R., Proc. Nat. Acad. Sci. U.S. 65, 1097 (1970). Doolittle, W. F. and Pace, N. R., Nature 228, 125 (1970). Brownlee, G. G., Sanger, F., and Barrell, B. G., J. Mol. Biol. 3,1, 379 (1968). Van Dijk-Salkinoja, M. S., Stoof, T. J., and Planta, R. J., Eur. J. Biochem. 12, 474 (1970). Ret61, J. and Planta, R. J., Eur. J. Biochem. 3, 248 (1967). Loening, U. E., Biochem. J. 113, 131 (1969). Richards, E. G., Coll, J. A., and Gratzer, W. B., Anal. Biochem. 12, 452 (1965). Sanger, F., Brownlee, G. G., and Barrell, B. G., J. Mol. Biol. 13, 373 (1965). Osawa, S., Ann. Rev. Biochem. 37, 109 (1968). Feunteun, J., Jordan, B. R., and Monier, R., J. Mol. Biol. 70, 465 (1972). Pace, N. R., Pato, M. L., McKibben, J., and Radcliffe, C. W., J. Mol. Biol. 75, 619 (1973).
249
OF BACTERIAL
RIBOSOMAL
RNA
T . J . STOOF AND R . J . PLANTA
Biochemisch Laboratorium, Vrije Universiteit, Amsterdam, The Netherlands
(Received 20 November, 1973) ABSTRACT. By comparison of the fingerprints of 5 S and 23 S ribosomal RNAs from Bacillus licheniformis with that of the precursor of 23 S ribosomal RNA, it can be shown that 5 S RNA is not a part of the precursor of 23 S ribosomal RNA. I. I N T R O D U C T I O N The genetic organization of the cistrons for bacterial ribosomal RNA species (23S, 16S and 5 S rRNA) has been studied in detail with Escherichia coli, Bacillus subtilis and B. megaterium [1-12]. These studies have shown that: (a) the number of gene copies for each RNA species is the same [1, 2], and (b) the 23 S, 16 S and 5 S rRNA genes are tightly linked and arranged as a block in the order 16 S-23 S-5 S [3-8]. Substantial evidence [5-8] has been obtained that each block of the linked three cistrons can be considered as a single 'transcriptional unit', in which transcription is initiated at the 16 S end and continues through the 23 S and 5 S RNA cistrons. Since no common RNA precursor of the three rRNA species has ever been found in bacteria, this model implies that the 16 S moiety is liberated from the growing RNA chain before transcription of the unit is complete. In fact, newly formed 16S and 23 S rRNAs are found first in precursor forms: the precursor form of 16S rRNA (designated as p l6S) is approximately 10~o larger than mature 16 S rRNA, and the precursor form of 23 S rRNA (p 23 S) is assumed to be about 200 nucleotides larger than mature 23 S rRNA [9-11 ]. Furthermore, it was inferred from several lines of indirect evidence that 5 SRNA is also transcribed as part of a rather large precursor molecule [4, 5, 7, 8, 12]. Taking these data together, it was tempting to propose that 5 S RNA, which in E. coli is only 120 nucleotides long [13], is part of p23S RNA, as was done by a number of authors [5, 7, 9, 11]. In this concept, the first transcription products from the bacterial ribosomal transcriptional unit are p l 6 S RNA and p23S RNA, after which 5S RNA is generated during the conversion of p23S R NA into 23S rRNA. We tested this hypothesis for B. licheniformis by comparing the fingerprints of 5 S, 23 S and p23S RNA with each other, and, as will be described in the present report, this led us to the conclusion that the 5 S RNA sequence is not present in the precursor of 23 S rRNA. II. MATERIALS A N D M E T H O D S Bacillus licheniformis (strain $244) was used in the present investigations. The bacteria were routinely grown at 37 ~ under vigorous aeration in a synthetic medium as previously described [14], or in a low-phosphate medium (see below). 243 Molecular Biology Reports 1 (1973) 243-249. All Rights Reserved Copyright 9 1973 by D. Reidel Publishing Company, Dordrecht-Holland
32p-labelled 23 S and 5 S rRNAs were obtained by adding 32p-orthophosphate to a final concentration of 0.05 mC ml- ~ to an early-exponential culture of B. licheniformis in a low-phosphatemedium (containing per litre: 6.05 g Tris-HC1, 50 mg MgSO4 97H20, 5 mg FeSO4, 2 g NH4C1, 1 g L-glutamic acid, 2 g KCI and 4.5 g standard bouillon, pH 7.3), continuing growth for 3 generations, collecting of cells by centrifugation at 3000 x g for l0 min, converting the cells to spheroplasts by treatment with lysozyme as described previously [14], and extracting RNA from the spheroplasts with phenol-SDS in essentially the same way as described by Ret 61 and Planta I15], except that the extraction was performed at 0 ~ The individual RNA species were separated by sucrose-gradient centrifugation in a Spinco SW 25-1 rotor at 22500 rpm for 16 hr [15]. The 23S rRNA was isolated from the appropriate fractions by ethanol-precipitation overnight at - 2 0 ~ and further purified by recentrifugation. As judged by electrophoretic analysis on 3~ polyacrylamide gels [l 6], the 23 S rRNA preparation thus obtained was practically homogeneous (contamination with 16 S rRNA was less than 2~). The 5 S RNA was purified by precipitation of the slowly sedimenting material from the appropriate gradient fractions with 3 vol of ethanol at - 2 0 ~ (overnight), and subsequent electrophoresis of the RNA sample on 10~ polyacrylamide gels [17]. The gels were cut in 1 mm slices, which were counted directly in a liquid scintillation counter. The slices containing the aEp-labelled 5 S RNA were pooled and eluted by shaking in a minimal volume of a solution of 1 M NaC1, 0.01 M Tris-HCl, pH 7.0 at 4 ~ overnight. p23 S RNA, pulse-labelled with 32p-orthophosphate, was prepared by adding to a culture in the mid-logarithmic growth phase on a low-phosphate medium (see above) chloramphenicol (final concentration 300 Ixgml- 1), and 3 min later 32p_orthophosphate (final concentration 0.5 mC ml- 1). The cells were harvested, converted to spheroplasts, and the RNA was extracted as described above, p23 S RNA was purified by repeated sucrose gradient centrifugation to a nearly homogeneous state, as judged by electrophoretic analysis on 3~ polyacrylamide gels [16]. p23S RNA, pulse-labelled with 3H-uridine was obtained in a similar manner, except that the culture was grown on a normal synthetic medium [14]. The fingerprints were made as follows: aliquots of 20 lag RNA in 0.005 ml 0.01 M Tris-HCl (pH 8.0) were digested with Tl-ribonuclease (enzyme-substrate ratio of l: 10) and alkaline phosphatase (enzyme-substrate ratio of 1:5) at 37 ~ for 30 min. The digestion products were separated in two dimensions by electrophoresis according to Sanger et al. [l 8]. The fingerprints were visualized by radioautography (medical X-ray film, type R, 3M Italia SPA). In one case (see Section III), fingerprinting was applied to a mixture of 32p., 14C_ and 3H-labelled RNAs. In this particular case, all a2p-containing spots (detected by radioautography) were cut out and counted for 3H and 14C after a nearly complete decay of the 32p-radioactivity. To this end, each spot was incubated with 0.5 ml Nuclear Chicago Solubilizer (NCS) in a counting vial at 37 ~ overnight. After addition of l0 ml toluene-based scintillation fluid, the vials were stored at 4 ~ until the 32p-activity had disappeared, and then counted in a liquid scintillation counter. III. RESULTS AND DISCUSSION Fig. 1 shows a typical electrophoretic separation pattern of B. licheniformis RNA, pulse-labelled with (~4C-methyl)-methionine, in the presence of chloramphenicol. It is well-known that inhibition of cellular protein synthesis (by chloramphenicol) results in an accumulation of the precursor forms of bacterial rRNA [19]. The pulse-labelled RNA appear to consist of well-defined RNA species with a clearly lower electrophoretic mobility than the corresponding mature rRNA components. That these rapidly-labelled RNA species do represent precursor forms of the rRNA components, may be inferred from the fact that they are methylated, and from the observation 244
20
10
23S
15
"'10 v
I
e9 I
O x c-
5
5
s
"T
/
/
I i f I I J J I I I I I I I J I I I I I
16S
5 1 v
I
ff O •
%
t'e9
,4.a
E U ,r E
E
c~ "o
"13 I
I
I
2O
4O
6O
FRACTION NUMBER Fig. 1. Polyacrylamide gel electrophoresis of pulse-labelled R N A of B. licheniformis. Exponentially growing cells in a n o r m a l synthetic m e d i u m [14] were labelled for three generations with 3H-uridine. Subsequently, chloramphenicol was added, 3 m i n later followed by pulse-labelling with (14C-methyl) methionine in the same way as described for saP-labelling in Section II. The cells were converted into spheroplasts, and the R N A was extracted as described in Section II. The R N A mixture was subjected to electrophoresis on 3 700 polyacrylamide gels, to which glycerol (7 % v/v) was added in order to facilitate slicing of the gels. The gels were sliced into 1 mm-slices with a Joyce-Loebl gel slicer, and subsequently monitored for all- and 14C-radioactivity after incubation overnight at 37 ~ in 10 ml toluene-based scintillation fluid containing the usual scintillator and 3 % N C S (Nuclear Chicago Solubilizer).
that they are processed to the mature rRNA components after removal of the antibiotic from the culture (results not shown). Similar separation patterns as in Fig. 1 were obtained after pulselabelling of the RNA with 3H-uridine or 32p-orthophosphate. In order to investigate the possible relationship between p23 S RNA and 5 S RNA, we decided to make fingerprints of both RNA species, and to look whether or not the fingerprint of p23S RNA contains spots characteristic of 5 S RNA. For the isolation of relatively pure p23 S RNA with sufficient high specific activity we preferred sucrose gradient centrifugation above gel electrophoresis. To our experience, elution of high-molecular-weight RNA from gel slices of low polyacrylamide concentrations produces RNA preparations highly contaminated with acrylamide. The presence of this contaminant in the RNA preparation results in fingerprints with a very bad resolution, particularly in the region of the larger oligonucleotides. Therefore, we subjected 32p-pulse-labelled RNA to sucrose gradient centrifugation. The radioactive fractions, sedimenting faster than 23 S rRNA were pooled, and this material was recentrifuged in the same way. 245
30
S
~0 20
D
X
d 10
I
I
20 40 FRACTION NUMBER
I
60
Fig. 2. Gel electrophoretic analysis of partially purified a~P-labelled p 23 S RNA. Labelling, extraction and purification of this precursor R N A are described in Section II, the conditions of electrophoresis are presented under Fig. 1.
Fig. 3. Two-dimensional separation patterns (upper parts) of a digest of a2p-labelled 5 S r R N A and 23 S rRNA, respectively. The R N A components, isolated as described in Section II, were digested with both Tl-ribonuclease and alkaline phosphatase. The removal of the 3'-terminal phosphate group enhances the mobility of the oligonucleotides in the second dimension which is important in particular for the larger fragments. In this group of fragments an unique spot from 5S R N A can be observed (indicated by an arrow). Faint spots are also visible in the fingerprint of 23 S rRNA, which are most probably due to an incomplete action of Tl-ribonuclease resulting in oligonucleotides with a terminal cyclic phosphate. For experimental details, see Section II.
246
Fig. 2 shows the electrophoretic analysis of an aliquot of the p23S R N A preparation thus obtained. It is obvious that the p23 S R N A preparation is still contaminated with a small amount of 23 S rRNA. However, this contamination does not really interfere with our approach, since we are looking for the presence of unique 5 S R N A spots, that is to say which are not present in a fingerprint of 23 S rRNA. Fig. 3 demonstrates that the rather simple fingerprint of 5 S R N A does indeed possess a spot (marked with an arrow), not coinciding with any spot of the 23 S rRNA-fingerprint (for the sake
Fig. 4. Two-dimensional separation patterns of a combined digest of 8~P-labelled 23 S plus 5 S rRNA, and p 23 S RNA, respectively. For further details see Section II and the legend to Fig. 3. 247
of clarity only the upper halves of the fingerprints are shown, i.e. the region where the larger oligonucleotides are located, and where differences might be expected to occur). However, for our purpose it is absolutely necessary that this oligonucleotide is an internal fragment, and does not correspond to a sequence containing the 5'- or 3'-end group. This argument is the more pertinent, since cleavage of a hypothetical common precursor of 5 S and 23 S rRNA seems not to be the final step in the 5S RNA maturation process [20, 21]. To settle this point, 32p-labelled 5S RNA was digested with Tl-ribonuclease alone to avoid the removal of a possible terminal phosphate group, the digest was fractionated according to Sanger et al. [18], and the spot corresponding to the oligonucleotide in question was located by radioautography. The oligonucleotide was eluted from the paper with 30% triethylamine carbonate (pH 10.0). After desalting of the sample by electrophoresis, the material was hydrolyzed with 1 N KOH for 16 hr at 37~ and the hydrolysis products were analyzed by electrophoresis on Whatman 3 MM at pH 3.5. By this type of analysis it could be established (as will be published elsewhere) that the oligonucleotide contains a 5'-OHand a -G-3'-P-end, and therefore represents an internal fragment of 5 S RNA with the bruto base
composition (C6U3G). In Fig. 4 the combined fingerprint of 32p-labelled 23 S plus 5 S rRNA can be compared with the fingerprint obtained from 32p-labelled p23S RNA. As can be seen, the unique 5S RNA spot, which is marked with an arrow in the combined fingerprint of 23 S plus 5 S rRNA, is absent in the fingerprint of p23S. Therefore, we conclude that p23S RNA does not contain the 5S RNA sequence. This conclusion was supported by the result of the following experiment. We isolated p23S RNA, pulse-labelled with 3H-uridine, and mixed with a fixed amount of 23S rRNA, uniformly labelled with 14C-uracil, and a small amount of 5 S RNA, uniformly labelled with 32p-orthophosphate. This mixture was digested and fingerprinted as described in the Methods section. The 32p-labelled oligonucleotides, derived from 5S RNA, were located by radioautography. All those spots were cut out, and after the decay of the 32p-radioactivity were assayed for an- and 14C-radioactivity as described in Section II. Although in the great majority of the spots both 3H- and 14C-counts were found, both activities were nearly completely absent in the unique 5S RNA-spot described above. This confirms the idea that this oligonucleotide, derived from 5 S RNA, is neither present in 23 S rRNA, nor in p 23 S RNA, in agreement with the conclusion drawn above. ACKNOWLEDGEMENTS The present investigations have been partly sponsored by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for the Advancement of Pure Research (ZWO). The authors highly appreciate the excellent technical support given by Miss V. C. H. F. de Regt. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 248
Smith, I., Dubnau, D., Morell, P., and Marmur, J., J. Mol. Biol. 33, 123 (1968). Pace, B. and Pace, N. R., J. Bacteriol. 105, 142 (1971). Colli, W. and Oishi, M., Proc. Nat. Acad. Sci. U.S. 64, 642 (1969). Bleyman, M., Kondo, M., Hecht, N., and Woese, C. R., J. Bacteriol. 99, 535 (1969). Pato, M. L. and von Meyenburg, K., ColdSpring Harbor Symp. Quant. Biol. 35, 497 (1970). Colli, W., Smith, I., and Oishi, M., J. Mol. Biol. 56, 117 (1971). Doolittle, W. F. and Pace, N. R., Proc. Nat. Acad. Sci. U.S. 68, 1786 (1971). Dennis, P. P. and Bremer, H., J. Mol. Biol. 75, 145 (1973). Hecht, N. B., Bleyman, M., and Woese, C. R., Proc. Nat. Acad. Sci. U.S. 59, 1278 (1968).
10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
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