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The Occurrence of Two Nicks in the Heavy Chloroplast Ribosomal RNA of Nicotiana rustica
Biochem. Physiol. Pflanzen 177, 431-439 (1982)
The Occurrence of Two Nicks in the Heavy Chloroplast Ribosomal RNA of N icotiana rustica SILVA LERBS and REINHOLD WOLLGIEHN Akademie der Wissenschaften der DDR, Forschungszentrum fiir Molekularbiologie und Medizin, Institut fiir Biochemie der Pflanzen Halle, Halle (Saale) Key Term Index: chloroplasts, ribosomal RNA, RNA synthesis, RNA processing and maturation;
Nl:cotiana rustica
Summary Newly synthesized 1.1 x 106 mol. wt. chloroplast ribosomal RNA molecules of Nicotiana rustica are unbroken polynucleotide chains. These molecules are stable during extraction and during electrophoresis under fully denaturing conditions in 6 M urea. During post-maturation of the 1.1 x 106 mol. wt. RNA two nicks are formed successively. At first the RNA is cleaved into two fragments with mol. weights of 0.7 and 0.4 x 106 , and in a second step the 0.7 x 106 mol. wt. fragment is further cleaved into two fragments of mol. weights 0.5 and 0.2 x 106• In older, fi-8 cm long leaves the newly synthesized heavy chloroplast rRNA remains more stable (at least 18 h) than in young, 2-3 cm long leaves where nicking begins already few hours after RNA synthesis.
Introduction
The newly synthesized mature 1.1 X 106 mol. weight chloroplast ribosomal RNA is composed of one unbroken polynucleotide chain. With time the RNA is cleaved at several particular points and can be separated into several fragments under denaturing conditions which fully destroy the secondary structure of the RNA (INGLE 1968; KOCHERT and SANSING 1971; LEAVER and INGLE 1971; LEAVER 1973; GRIERSON 1974; MUNSCHE and WOLLGIEHN 1974; ROSNER et al. 1974; MACHE et al. 1978; SPEIRS and GRIERSON 1978; LOISEAUX et al. 1979). Under non-denaturing conditions the fragmentation of the RNA is prevented by non-covalent interactions in the secondary structure of the molecule. The presence of magnesium is one of the important pre-requisites to stabilize the RNA. In a previous paper (MUNSCHE and WOLLGIEHN 1974) we have shown that chloroplast 1.1 x 106 mol. wt. rRNA of Nicotiana rustica is dissociated into fragments of distinct molecular weights if denatured by heating or treatment with urea or dimethylsulfoxide. Ribosomal precursor RNA and newly synthesized 1.1 X 106 mol. wt. rRNA is stable under these conditions. In our early experiments we have used 1.1 X 106 mol. wt. rRNA which was contaminated with other kinds of RNA. Therefore, we repeated the experiments with pure 1.1 X 106 mol. wt. RNA. The results have shown that this chloroplast rRNA of Nicotiana rustica contains two labile sites and is successively cleaved into three fragments. The rate of cleavage depends on the age of the ribosomes and the age of the leaves. 28 Biochem. Physiol., Pflanzen, Bd. 177
432
S. LERBs and R. W OLLGIEHN
Material and Methods All experiments were carried out with greenhouse-cultivated plants of Nicotiana rustica. Leaves of different age were taken from the same type of growing plants. Incubation of leaf material with 32P, isolation of chloroplasts and purification by flotation in sucrose, extraction of nucleic acids and fractionation by polyacrylamide gel electrophoresis were carried out as already described (MUNSCHE and WOLLGIEHN 1973; W OLLGIEHN et aI1978). Isolation of crude chloroplast ribosomes: Leaves were ground in homogenization medium (100 mM Tris-HCl, pH 7.5; 50 mM KCl; 5 mM MgCI 2 ; 500 mM sucrose; 0.1 % p-aminosalicylic acid), filtered through 6 layers of cheesecloth and centrifuged at 500 . g for 5 min to remove cell debris and nuclei. Chloroplasts were pelleted from the supernatant by centrifugation 30 s at 5000 . g, The chloroplast pellet was suspended in 10 ml of homogenization medium without sucrose and p-aminosalycylic acid and gently stirred at 2 °C for 10 min. Lysed chloroplasts were centrifuged et 10,000 . g for 30 min to separate free (in the supernatant) and membrane-bound ribosomes. RNA was extracted from the two fractions.
Results
Chloroplasts of growing 6-8 cm long leaves of Nicotiana rustica were isolated and purified by flotation in 1. 7 M sucrose and their RNA was separated by gel electrophoresis (Fig. 1, A). The part of the gel containing the 1.1 X 106 mol. wt. rRNA (dotted region)
A
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at 25"C
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Fig. 1. Fragmentation of chloroplast 1.1 x 106 mol. wt. rRN A in dependence on the temperature. Chloroplast nucleic acids extracted from 6---8 cm long leaves were separated by electrophoresis on 2.4 % polyacrylamide gels at 10°C (A). After monitoring at 260 nm the gel section containing the 1.1 x 106 RNA was cut out and immersed into 100 ttl of 100 mM Tris-HCI, pH 7.5, 50 111M KCl, and kept at elevated temperatures. The gel slices were layered on the top of step gels consisting of 3 and 2.4% acrylamide (aa). After reelectrophoresis the optical density was scanned again at 260 nm (B and C).
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Fig. 2. Reelectrophoresis of 1.1 x 10 6 mol. wt. rRNA cleavage products (obtained by heating the RNA) after complete denaturation the fragments in 6 111 urea. 1.1 x 106 rRNA and its e1eavage products (after heating) were obtained as in Fig. 1. The gel slices with four cleavage products were heated ag11in in the presence of 6 l\i urea before reelectrophoresis on a step gel in the presenee of urea.
was immersed into 100 fll of buffer and either kept at room temperature for 30 min or additionally heated for 1 min to 60°C. The gel slice was layered on the top of a step gel for re-electrophoresis. Figs. IB and C show that the 1.1 X 106 mol. wt. rRNA is partially fragmented even when it is kept for some time at room temperature. Five breakdown fragments were found with molecular weights of 0.9, 0.7,0.5,0.4 and 0.2 X 106 (Fig. IB). This is in good agreement with results from LEAVER and INGLE (1971); GRIERSON (1974); MUNSCHE and WOLLGIEH:\, (1974) and MACHE et al. (1978) obtained with 1.1 X 106 rRNA from different plants. After heating the rRNA to 60°C a much larger part of the RNA is cleaved and accordingly the amounts of the cleavage products increase except that of the 0.9 X 106 mol. wt. fragment (Fig. lC). During a second heating of the gel slice containing only the 0.9 X 106 mol. wt. RNA for 1 min to 60°C in 6 M urea instead of buffer to eliminate completely the secondary structure of the RNA this RNA fragment is completely cleaved into two fragments of mol. wt. 0.5 and 0.4 X 106 • This means the 0.9 X 106 RNA fragment results from incomplete denaturation of the 1.1 X 106 mol. wt. rRNA at 60°C in Tris-HCI buffer, pH 7.5. In contrast, the 0.7,0.5 and 0.4 X 106 mol. wt. fragments are stable under these conditions (Fig. 2). The 0.2 X 106 fragment and thc remaining 1.1 X 106 mol. wt. rRNA are also not further cleaved in 6 M urea (data not shown). These molecules which are stable in urea are unbroken polynucleotide chains without any additional nicks. 28*
434
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1 min
and R. WOLLGIEHN
(Gradient gel)
at
11 RNA
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- - - E/ectrophoretic mobility Fig. 3. Electrophoresis of 1.1 x 106 mol. wt. rRN A fragments obtained by heating in a gradient gel to detect low molecular weight fragments. The gel consisted of a gradient of 2.2-7 % acrylamide. A: Total leaf RNA as control (after heating). B: Chloroplast 1.1 x 106 mol. wt. rRNA from young 2-3 em long leaves after labelling with 32P. The 1.1 x 1()6 mol. wt. rRNA was obtained by gel electrophoresis and heated to 60 DC before reelectrophoresis on a gradient gel. After monitoring the optical density at 260 nm the gel was dried on filter paper before autoradiography. The alltoradiographs were scanned at 600 nm.
In a further experiments we have checked whether additional fragments with molecular weights smaller than 0.2 x 106 occur beside the four fragments determined above. For this purpose we have used a polyacrylamide gradient gel that separates RNA from 1.3 x 106 mol. wt. up to RNA smaller than tRNA (Fig. 3, total RNA). The 32p labelled 1.1 x 106 rRNA was denatured in Tris-HCI buffer, pH 7.5 at 60 DC for 1 min (or in 6 M urea) and analysed with this gel system (Fig. 3, 1.1 x 106 RNA). Neither "older" (optical density) nor newly synthesized (radioactivity) rRNA results in fragments with mol. weights between 0.2 X 106 and tRNA. From the results hitherto described one might conclude that three types of 1.1 X 106 mol. wt. rRNA exist within the chloroplasts of Nicotiana rustica: Unbroken polynucleotide chains and two types of nicked (post-matured) molecules. The first type contains one break and splits into two fragments of 0.7 and 0.4 X 106 mol. wt. and the second type contains two breaks and yealds three fragments of 0.5, 0.4 and 0.2 X 106 mol. wt. under denaturing conditions. The two types of post-matured RNA could either reflect a sequence in the formation of two breaks in the same RNA molecule during post-maturation, or they may correspond to two different types of ribosomes, e.g. membrane-bound or free ribosomes. To check the second possibility we isolated two
Nicked Chloroplast Ribosomal RNA 18h 32p
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05
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crude chloroplast fractions containing free or membrane-bound ribosomes (see material and methods). The fragments of the 1.1 X 106 mol. wt. rRNA of these two kinds of ribosomes were analysed as in Fig. 1, but the two types showed the same fragmentation profile as presented in Fig. le. Therefore, this result does not provide evidence for the localization of the differently nicked chloroplast r RNAs in different kinds of ribosomes. In order to proof the possibility of a sequence in the formation of the two nicks in the RNA molecule it was necessary to find experimental conditions that allow the examination of the fragmentation of newly synthesized, radioactively labelled RNA. As already shown by MUNSCHE and WOLLGIEHN (1974) in experiments with isolated leaf material and intact rooted leaves, pulse-labelled mature rRNA is stable during heat treatment. The 1.1 X 106 mol. wt. rRNA contains the characteristic breaks only a few days after its labelling. On the other hand, 32p incubation of isolated leaves can be done only for a limited time (not more than 24 h). Therefore we tried to find suitable material for the further experiments in which post-maturation of the RNA can be observed already a few hours after labelling the rRNA. In preceding experiments we have found that the kinetics of synthesis and processing of chloroplast rRNA markedly differs in leaves of different age. These processes are retarded in old leaves (WOLLGIEHN et al. 1976). It seemed to be likrly that also post-maturation of chloroplast r RNA differs in young and older leaves according to the results from bGLE (1968) and ROSNER et al. (1974), who also found differences in the rate of chloroplast rRNA nicking in dependence on the physiological state of the plant material. We examined the fragmentation of newly synthesized 1.1 X ]06 mol. wt. rRNA isolated from very young leaves (2-3 cm long) and from older, still growing leaves (6-8 cm long) after 18 h labelling isolated leaves with 32P. The fragmentation patterns are shown in Fig. 4. From the older leaves only a very small part of the newly synthesized, labelled R~A is already nicked (Fig. 4B). In contrast, in vrry young leaves more than half of the labelled 1.1 X 106 mol. wt. rRNA molecules are nicked after the same time of labelling (Fig. 4A, radioactivity). Thus, very young leaves provide a useful material to
s. LERBS and R. WOLLGIEIlX
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study the kinetics of the formation of "hidden breaks" in the polynucleotide chain of the heavy chloroplast rRXA. As expected, the non-Iabellcd "old" 1.1 X 106 mol. wt. rRNA is nicked in nearly the same way in alllcaves, independent of their agc. Whcn thc isolated young lcavcs were incubated with 32p for different time (5-24 h) the patterns of the labelled fragmentation products of the 1.1 X 106 mol. wt. RNA
Nicked Chloroplast Ribosomal RNA
437
change in a characteristic way (Fig. 5). After 5 h labelling a great proportion of the RNA was intact after denaturation. The molecular weights of the two main fragments found in nearly equal amounts were 0.7 and 0.4 X 106 • Only small amounts of 0.5 and 0.2 x 106 mol. wt. RNA (the latter was not detected by the electrophoretic conditions chosen for the experiment shown in Fig. 5), but also 0.9 x 106 mol. wt. RNA were found. This means the newly synthesized 1.1 X 106 mol. wt. rRNA after a few hours contains mainly one labile site, leading to two fragments of 0.7 and 0.4 x 106 mol. wt. Only some of the newly synthesized rRNA molecules already contain a second break which leads to additional fragments of 0.5 and 0.2 X 106 mol. wt., but also to some 0.9 X 106 mol. wt. RNA as a consequence of incomplete cleavage during heating. After a longer time of 32p incubation the amount of stable labelled 1.1 X 106 mol. wt. RNA is reduced, nearly quantitative after 24 h of labelling. The 0.7 X 106 mol. wt. fragment is also reduced relative to 0.4 X 106 mol. wt. RNA and simultaneously increased the 0.5 X 106 (and 0.2 x 106) mol. wt. fragment, This means that during post-maturation of 1.1 x 106 mol. wt. rRNA of Nicotiana rustica two nicks are formed successively leading to three fragments of 0.5, 0.4 and 0.2 X 106 mol. wt. after denaturation. Discussion
The occurence of "hidden breaks" as a result of post-maturation processes is a property of ribosomal RNAs of many organisms. In chloroplasts the 0.56 X 106 mol. wt. ribosomal RNA is stable, whereas the 1.1 x 106 mol. wt. rRNA of most plants contains several breaks, leading to fragmentation of this RNA under denaturing conditions. The size and the number of the fragments produced from 1.1 X 106 mol. wt. RNA varies with different plant species (LEAVER and INGLE 1971). To obtain a clear picture about the cleavage pattern of the 1.1 x 106 mol. wt. rRNA of Nicotiana rustica we have re-investigated our previous experiments (MUNSCHE and WOLLGIEHN 1974) using pure 1.1 X 106 mol. wt. rRNA isolated by polyacrylamide gel electrophoresis. Our results confirm data from different laboratories with different plant material that the newly synthesized 1.1 X 106 mol. wt. rRNA within the newly assembled ribosomes is intact and with time the RNA is cleaved at specific sites (INGLE 1968; LEAVER and INGLE 1971; GRIERSON 1974; MUNSCHE and WOLLGIEHN 1974; ROSNER et al. 1974; MACHE et al. 1978; LOISEAUX et al. 1979). The proportion of the total 1.1 x 106 mol. wt. RNA molecules that are fragmented varies with the physiological state and with the age of the tissue (INGLE 1968; ROSNER at al. 1974). In Nicotiana rnstica the rate of nicking is greater in very young than in older leaves. This corresponds to a higher rate of synthesis and processing of the chloroplast ribosomal RNA in young leaves in comparison to mature and old leaves (WOLLGIEHN et al. 1976). Our experiments have further shown that the 1.1 X 106 mol. wt. RNA of Nicotiana rustica is cleaved at two specific sites and that cleavage at this two sites occurs successively during post-maturation of the ribosomes (Fig. 5 and 6). Discrimination between these two steps is not absolute, but during first phase of post-maturation cleavage leads mainly to fragments of 0.7 a11d 0.4 x 106 mol. wt. and later to 0.5, 0.4 and 0.2 X 106
438
S. LERBS and R. WOLLGIEHN 11 x106 rRNA
First cleavage 0.1 0.2
I
0.4
Second cleavage 05
0.4
Fig. 6. Scheme of the post-maturational cleavage of chloroplast 1.1 x 106 mol. wt. rRNA. of Nicotiana rustica. Approximate molecular weights of the cleavage products.
mol. wt. Another fragment of 0.9 x 106 mol. wt. which is found after heating the RNA or in the absence of magnesium is not an intact polynucleotide. It can be further separated into two fragments under conditions of complete denaturation the RNA (6 M urea). The 0.9 x 106 mol. wt. fragment only occurs, because the second break within the RNA is more labile than the first one. We did not find any data for the argument that the two kinds of RNA containing one or two nicks belong to different types of ribosomes, i.e. free or membrane-bound chloroplast ribosomes. WRESCHNER et al. (1978) described the occurence of a membrane-bound factor in reticulocytes, which causes a specific cleavage of the 28 S rRNA correlating with an inhibition of cell-free protein synthesis. Differences in the physiological state or in the age of the plant material give additional complications. Our experiments have shown that such different conditions only lead to changes in the rate of cleavage but not to changes in number and size of the fragments. It is possible that many of the differences found in different labotatories concerning number and size of the 1.1 x 106 mol. wt. RNA fragments could be brought into correspondence, if in all experiments the same conditions (e.g. complete denaturation) were used. The biological function of the "hidden breaks" within some kinds of ribosomal RNA remains further unknown. References GRIERSON, D.: Characterization of ribonucleic acid components from leaves of Phaseolus aureus. Europ. J. Biochem. 44,509-515 (1974). INGLE, J.: Synthesis and stability of chloroplast ribosomal RNAs. Plant Physiol. 43, 1448-1454
(1968). KOCHERT, G., and SANSING, N.: Isolation and characterization of nucleic acids from Volvox carterii. Biochim. Biophys. Acta 238, 397-405 (1971). LEAVER, C.: Molecular integrity of chloroplast ribonucleic acid. Biochem. J. 130, 237-240 (1973). LEAVER, C. J., and INGLE, J.: The molecular integrity of chloroplast ribosomal ribonucleic acid. Biochem. J. 123, 235-243 (1971). LorSEAux, S., MACHE, R., and RAZIER, C.: Heterogeneity of the 23 S ribosomal RNA in pheoplasts of Pylaiella littoralis (L.) Kjelm., Phaeophyta. Physiol. Veg. 17, 619-629 (1979). MAcHE, R., JALLIFFIER-VERNE, M., ROZIER, C., and LOISEAUX, S.: Molecular weight determination of precursor, mature and post-mature plastid ribosomal RNA from spinach using fully denaturing conditions. Biochim. Biophys. Acta 017,390-399 (1978).
Nicked Chloroplast Ribosomal RNA
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MUNSCHE, D., and WOLLGIEHN, R.: Die Synthese von ribosomaler RNA in Chloroplasten von Nicotiana rustica. Biochim. Biophys. Acta 294, 106-117 (1973). MUNSCHE, D., and WOLLGIEHN, R.: Altersabhiingige Labilitat der ribosomalen RNA aus Chloroplasten von Nicotiana rustica. Biochim. Biophys. Acta 340, 437-445 (1974). ROSNER, A., PORATH, D., and GRESSEL, J.: The distribution of plastid ribosomes and the integrity of plastid ribosomal RNA during the greening and maturation of Spirodela fronds. Plant and Cell Physiol. Hi, 891-902 (1974). SPEIRS, J., and GRIERSON, D.: Isolation and characterisation of 14-S RNA from spinach chloroplasts. Biochim. Biophys. Acta 1)21, 619-633 (1978). WRESCHNER, D., MELLOUL, D., and HERZBERG, M.: Interaction between membrane functions and protein synthesis in reticulocytes: Specific Cleavage of 28-S ribosomal RNA by a membrane constituent. Eur. J. Biochem. 81), 233-240 (1978). WOLLGIEHN, R., LERBS, S., and MUNSCHE, D.: Synthesis of ribosomal RNA in chloroplasts from tobacco leaves of different age. Biochem. Physiol. Pflanzen 170, 381-387 (1976). WOLLGIEHN, R., LERBS, S., and MUNSCHE, D.: Eigenschaften einer Chloroplasten-RNA vom Molekulargewicht 0.5 x 106 aus Nicotiana rustica. Bioehem. Physiol. Pflanzen 173, 60-69 (1978).
Received December 29, 1981 Authors' address: Dr. SILVA LERBS and Dr. REINHOLD WOLLGIEHN, Akademie der Wissenschaften der DDR, Forschungszentrum fUr Molekularbiologie und Medizin, Institut fur Biochemie der Pflanzen Halle (Saale), Weinberg 3, DDR - 4020 Halle (Saale).
The Occurrence of Two Nicks in the Heavy Chloroplast Ribosomal RNA of N icotiana rustica SILVA LERBS and REINHOLD WOLLGIEHN Akademie der Wissenschaften der DDR, Forschungszentrum fiir Molekularbiologie und Medizin, Institut fiir Biochemie der Pflanzen Halle, Halle (Saale) Key Term Index: chloroplasts, ribosomal RNA, RNA synthesis, RNA processing and maturation;
Nl:cotiana rustica
Summary Newly synthesized 1.1 x 106 mol. wt. chloroplast ribosomal RNA molecules of Nicotiana rustica are unbroken polynucleotide chains. These molecules are stable during extraction and during electrophoresis under fully denaturing conditions in 6 M urea. During post-maturation of the 1.1 x 106 mol. wt. RNA two nicks are formed successively. At first the RNA is cleaved into two fragments with mol. weights of 0.7 and 0.4 x 106 , and in a second step the 0.7 x 106 mol. wt. fragment is further cleaved into two fragments of mol. weights 0.5 and 0.2 x 106• In older, fi-8 cm long leaves the newly synthesized heavy chloroplast rRNA remains more stable (at least 18 h) than in young, 2-3 cm long leaves where nicking begins already few hours after RNA synthesis.
Introduction
The newly synthesized mature 1.1 X 106 mol. weight chloroplast ribosomal RNA is composed of one unbroken polynucleotide chain. With time the RNA is cleaved at several particular points and can be separated into several fragments under denaturing conditions which fully destroy the secondary structure of the RNA (INGLE 1968; KOCHERT and SANSING 1971; LEAVER and INGLE 1971; LEAVER 1973; GRIERSON 1974; MUNSCHE and WOLLGIEHN 1974; ROSNER et al. 1974; MACHE et al. 1978; SPEIRS and GRIERSON 1978; LOISEAUX et al. 1979). Under non-denaturing conditions the fragmentation of the RNA is prevented by non-covalent interactions in the secondary structure of the molecule. The presence of magnesium is one of the important pre-requisites to stabilize the RNA. In a previous paper (MUNSCHE and WOLLGIEHN 1974) we have shown that chloroplast 1.1 x 106 mol. wt. rRNA of Nicotiana rustica is dissociated into fragments of distinct molecular weights if denatured by heating or treatment with urea or dimethylsulfoxide. Ribosomal precursor RNA and newly synthesized 1.1 X 106 mol. wt. rRNA is stable under these conditions. In our early experiments we have used 1.1 X 106 mol. wt. rRNA which was contaminated with other kinds of RNA. Therefore, we repeated the experiments with pure 1.1 X 106 mol. wt. RNA. The results have shown that this chloroplast rRNA of Nicotiana rustica contains two labile sites and is successively cleaved into three fragments. The rate of cleavage depends on the age of the ribosomes and the age of the leaves. 28 Biochem. Physiol., Pflanzen, Bd. 177
432
S. LERBs and R. W OLLGIEHN
Material and Methods All experiments were carried out with greenhouse-cultivated plants of Nicotiana rustica. Leaves of different age were taken from the same type of growing plants. Incubation of leaf material with 32P, isolation of chloroplasts and purification by flotation in sucrose, extraction of nucleic acids and fractionation by polyacrylamide gel electrophoresis were carried out as already described (MUNSCHE and WOLLGIEHN 1973; W OLLGIEHN et aI1978). Isolation of crude chloroplast ribosomes: Leaves were ground in homogenization medium (100 mM Tris-HCl, pH 7.5; 50 mM KCl; 5 mM MgCI 2 ; 500 mM sucrose; 0.1 % p-aminosalicylic acid), filtered through 6 layers of cheesecloth and centrifuged at 500 . g for 5 min to remove cell debris and nuclei. Chloroplasts were pelleted from the supernatant by centrifugation 30 s at 5000 . g, The chloroplast pellet was suspended in 10 ml of homogenization medium without sucrose and p-aminosalycylic acid and gently stirred at 2 °C for 10 min. Lysed chloroplasts were centrifuged et 10,000 . g for 30 min to separate free (in the supernatant) and membrane-bound ribosomes. RNA was extracted from the two fractions.
Results
Chloroplasts of growing 6-8 cm long leaves of Nicotiana rustica were isolated and purified by flotation in 1. 7 M sucrose and their RNA was separated by gel electrophoresis (Fig. 1, A). The part of the gel containing the 1.1 X 106 mol. wt. rRNA (dotted region)
A
c
B , 30 min
at 25"C
056 11
as 07
02
-
Dectrophoretlc mob/lity
Fig. 1. Fragmentation of chloroplast 1.1 x 106 mol. wt. rRN A in dependence on the temperature. Chloroplast nucleic acids extracted from 6---8 cm long leaves were separated by electrophoresis on 2.4 % polyacrylamide gels at 10°C (A). After monitoring at 260 nm the gel section containing the 1.1 x 106 RNA was cut out and immersed into 100 ttl of 100 mM Tris-HCI, pH 7.5, 50 111M KCl, and kept at elevated temperatures. The gel slices were layered on the top of step gels consisting of 3 and 2.4% acrylamide (aa). After reelectrophoresis the optical density was scanned again at 260 nm (B and C).
433
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was immersed into 100 fll of buffer and either kept at room temperature for 30 min or additionally heated for 1 min to 60°C. The gel slice was layered on the top of a step gel for re-electrophoresis. Figs. IB and C show that the 1.1 X 106 mol. wt. rRNA is partially fragmented even when it is kept for some time at room temperature. Five breakdown fragments were found with molecular weights of 0.9, 0.7,0.5,0.4 and 0.2 X 106 (Fig. IB). This is in good agreement with results from LEAVER and INGLE (1971); GRIERSON (1974); MUNSCHE and WOLLGIEH:\, (1974) and MACHE et al. (1978) obtained with 1.1 X 106 rRNA from different plants. After heating the rRNA to 60°C a much larger part of the RNA is cleaved and accordingly the amounts of the cleavage products increase except that of the 0.9 X 106 mol. wt. fragment (Fig. lC). During a second heating of the gel slice containing only the 0.9 X 106 mol. wt. RNA for 1 min to 60°C in 6 M urea instead of buffer to eliminate completely the secondary structure of the RNA this RNA fragment is completely cleaved into two fragments of mol. wt. 0.5 and 0.4 X 106 • This means the 0.9 X 106 RNA fragment results from incomplete denaturation of the 1.1 X 106 mol. wt. rRNA at 60°C in Tris-HCI buffer, pH 7.5. In contrast, the 0.7,0.5 and 0.4 X 106 mol. wt. fragments are stable under these conditions (Fig. 2). The 0.2 X 106 fragment and thc remaining 1.1 X 106 mol. wt. rRNA are also not further cleaved in 6 M urea (data not shown). These molecules which are stable in urea are unbroken polynucleotide chains without any additional nicks. 28*
434
S.
LE](BS
1 min
and R. WOLLGIEHN
(Gradient gel)
at
11 RNA
Tocal RNA
0.2
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- - - E/ectrophoretic mobility Fig. 3. Electrophoresis of 1.1 x 106 mol. wt. rRN A fragments obtained by heating in a gradient gel to detect low molecular weight fragments. The gel consisted of a gradient of 2.2-7 % acrylamide. A: Total leaf RNA as control (after heating). B: Chloroplast 1.1 x 106 mol. wt. rRNA from young 2-3 em long leaves after labelling with 32P. The 1.1 x 1()6 mol. wt. rRNA was obtained by gel electrophoresis and heated to 60 DC before reelectrophoresis on a gradient gel. After monitoring the optical density at 260 nm the gel was dried on filter paper before autoradiography. The alltoradiographs were scanned at 600 nm.
In a further experiments we have checked whether additional fragments with molecular weights smaller than 0.2 x 106 occur beside the four fragments determined above. For this purpose we have used a polyacrylamide gradient gel that separates RNA from 1.3 x 106 mol. wt. up to RNA smaller than tRNA (Fig. 3, total RNA). The 32p labelled 1.1 x 106 rRNA was denatured in Tris-HCI buffer, pH 7.5 at 60 DC for 1 min (or in 6 M urea) and analysed with this gel system (Fig. 3, 1.1 x 106 RNA). Neither "older" (optical density) nor newly synthesized (radioactivity) rRNA results in fragments with mol. weights between 0.2 X 106 and tRNA. From the results hitherto described one might conclude that three types of 1.1 X 106 mol. wt. rRNA exist within the chloroplasts of Nicotiana rustica: Unbroken polynucleotide chains and two types of nicked (post-matured) molecules. The first type contains one break and splits into two fragments of 0.7 and 0.4 X 106 mol. wt. and the second type contains two breaks and yealds three fragments of 0.5, 0.4 and 0.2 X 106 mol. wt. under denaturing conditions. The two types of post-matured RNA could either reflect a sequence in the formation of two breaks in the same RNA molecule during post-maturation, or they may correspond to two different types of ribosomes, e.g. membrane-bound or free ribosomes. To check the second possibility we isolated two
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crude chloroplast fractions containing free or membrane-bound ribosomes (see material and methods). The fragments of the 1.1 X 106 mol. wt. rRNA of these two kinds of ribosomes were analysed as in Fig. 1, but the two types showed the same fragmentation profile as presented in Fig. le. Therefore, this result does not provide evidence for the localization of the differently nicked chloroplast r RNAs in different kinds of ribosomes. In order to proof the possibility of a sequence in the formation of the two nicks in the RNA molecule it was necessary to find experimental conditions that allow the examination of the fragmentation of newly synthesized, radioactively labelled RNA. As already shown by MUNSCHE and WOLLGIEHN (1974) in experiments with isolated leaf material and intact rooted leaves, pulse-labelled mature rRNA is stable during heat treatment. The 1.1 X 106 mol. wt. rRNA contains the characteristic breaks only a few days after its labelling. On the other hand, 32p incubation of isolated leaves can be done only for a limited time (not more than 24 h). Therefore we tried to find suitable material for the further experiments in which post-maturation of the RNA can be observed already a few hours after labelling the rRNA. In preceding experiments we have found that the kinetics of synthesis and processing of chloroplast rRNA markedly differs in leaves of different age. These processes are retarded in old leaves (WOLLGIEHN et al. 1976). It seemed to be likrly that also post-maturation of chloroplast r RNA differs in young and older leaves according to the results from bGLE (1968) and ROSNER et al. (1974), who also found differences in the rate of chloroplast rRNA nicking in dependence on the physiological state of the plant material. We examined the fragmentation of newly synthesized 1.1 X ]06 mol. wt. rRNA isolated from very young leaves (2-3 cm long) and from older, still growing leaves (6-8 cm long) after 18 h labelling isolated leaves with 32P. The fragmentation patterns are shown in Fig. 4. From the older leaves only a very small part of the newly synthesized, labelled R~A is already nicked (Fig. 4B). In contrast, in vrry young leaves more than half of the labelled 1.1 X 106 mol. wt. rRNA molecules are nicked after the same time of labelling (Fig. 4A, radioactivity). Thus, very young leaves provide a useful material to
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study the kinetics of the formation of "hidden breaks" in the polynucleotide chain of the heavy chloroplast rRXA. As expected, the non-Iabellcd "old" 1.1 X 106 mol. wt. rRNA is nicked in nearly the same way in alllcaves, independent of their agc. Whcn thc isolated young lcavcs were incubated with 32p for different time (5-24 h) the patterns of the labelled fragmentation products of the 1.1 X 106 mol. wt. RNA
Nicked Chloroplast Ribosomal RNA
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change in a characteristic way (Fig. 5). After 5 h labelling a great proportion of the RNA was intact after denaturation. The molecular weights of the two main fragments found in nearly equal amounts were 0.7 and 0.4 X 106 • Only small amounts of 0.5 and 0.2 x 106 mol. wt. RNA (the latter was not detected by the electrophoretic conditions chosen for the experiment shown in Fig. 5), but also 0.9 x 106 mol. wt. RNA were found. This means the newly synthesized 1.1 X 106 mol. wt. rRNA after a few hours contains mainly one labile site, leading to two fragments of 0.7 and 0.4 x 106 mol. wt. Only some of the newly synthesized rRNA molecules already contain a second break which leads to additional fragments of 0.5 and 0.2 X 106 mol. wt., but also to some 0.9 X 106 mol. wt. RNA as a consequence of incomplete cleavage during heating. After a longer time of 32p incubation the amount of stable labelled 1.1 X 106 mol. wt. RNA is reduced, nearly quantitative after 24 h of labelling. The 0.7 X 106 mol. wt. fragment is also reduced relative to 0.4 X 106 mol. wt. RNA and simultaneously increased the 0.5 X 106 (and 0.2 x 106) mol. wt. fragment, This means that during post-maturation of 1.1 x 106 mol. wt. rRNA of Nicotiana rustica two nicks are formed successively leading to three fragments of 0.5, 0.4 and 0.2 X 106 mol. wt. after denaturation. Discussion
The occurence of "hidden breaks" as a result of post-maturation processes is a property of ribosomal RNAs of many organisms. In chloroplasts the 0.56 X 106 mol. wt. ribosomal RNA is stable, whereas the 1.1 x 106 mol. wt. rRNA of most plants contains several breaks, leading to fragmentation of this RNA under denaturing conditions. The size and the number of the fragments produced from 1.1 X 106 mol. wt. RNA varies with different plant species (LEAVER and INGLE 1971). To obtain a clear picture about the cleavage pattern of the 1.1 x 106 mol. wt. rRNA of Nicotiana rustica we have re-investigated our previous experiments (MUNSCHE and WOLLGIEHN 1974) using pure 1.1 X 106 mol. wt. rRNA isolated by polyacrylamide gel electrophoresis. Our results confirm data from different laboratories with different plant material that the newly synthesized 1.1 X 106 mol. wt. rRNA within the newly assembled ribosomes is intact and with time the RNA is cleaved at specific sites (INGLE 1968; LEAVER and INGLE 1971; GRIERSON 1974; MUNSCHE and WOLLGIEHN 1974; ROSNER et al. 1974; MACHE et al. 1978; LOISEAUX et al. 1979). The proportion of the total 1.1 x 106 mol. wt. RNA molecules that are fragmented varies with the physiological state and with the age of the tissue (INGLE 1968; ROSNER at al. 1974). In Nicotiana rnstica the rate of nicking is greater in very young than in older leaves. This corresponds to a higher rate of synthesis and processing of the chloroplast ribosomal RNA in young leaves in comparison to mature and old leaves (WOLLGIEHN et al. 1976). Our experiments have further shown that the 1.1 X 106 mol. wt. RNA of Nicotiana rustica is cleaved at two specific sites and that cleavage at this two sites occurs successively during post-maturation of the ribosomes (Fig. 5 and 6). Discrimination between these two steps is not absolute, but during first phase of post-maturation cleavage leads mainly to fragments of 0.7 a11d 0.4 x 106 mol. wt. and later to 0.5, 0.4 and 0.2 X 106
438
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Fig. 6. Scheme of the post-maturational cleavage of chloroplast 1.1 x 106 mol. wt. rRNA. of Nicotiana rustica. Approximate molecular weights of the cleavage products.
mol. wt. Another fragment of 0.9 x 106 mol. wt. which is found after heating the RNA or in the absence of magnesium is not an intact polynucleotide. It can be further separated into two fragments under conditions of complete denaturation the RNA (6 M urea). The 0.9 x 106 mol. wt. fragment only occurs, because the second break within the RNA is more labile than the first one. We did not find any data for the argument that the two kinds of RNA containing one or two nicks belong to different types of ribosomes, i.e. free or membrane-bound chloroplast ribosomes. WRESCHNER et al. (1978) described the occurence of a membrane-bound factor in reticulocytes, which causes a specific cleavage of the 28 S rRNA correlating with an inhibition of cell-free protein synthesis. Differences in the physiological state or in the age of the plant material give additional complications. Our experiments have shown that such different conditions only lead to changes in the rate of cleavage but not to changes in number and size of the fragments. It is possible that many of the differences found in different labotatories concerning number and size of the 1.1 x 106 mol. wt. RNA fragments could be brought into correspondence, if in all experiments the same conditions (e.g. complete denaturation) were used. The biological function of the "hidden breaks" within some kinds of ribosomal RNA remains further unknown. References GRIERSON, D.: Characterization of ribonucleic acid components from leaves of Phaseolus aureus. Europ. J. Biochem. 44,509-515 (1974). INGLE, J.: Synthesis and stability of chloroplast ribosomal RNAs. Plant Physiol. 43, 1448-1454
(1968). KOCHERT, G., and SANSING, N.: Isolation and characterization of nucleic acids from Volvox carterii. Biochim. Biophys. Acta 238, 397-405 (1971). LEAVER, C.: Molecular integrity of chloroplast ribonucleic acid. Biochem. J. 130, 237-240 (1973). LEAVER, C. J., and INGLE, J.: The molecular integrity of chloroplast ribosomal ribonucleic acid. Biochem. J. 123, 235-243 (1971). LorSEAux, S., MACHE, R., and RAZIER, C.: Heterogeneity of the 23 S ribosomal RNA in pheoplasts of Pylaiella littoralis (L.) Kjelm., Phaeophyta. Physiol. Veg. 17, 619-629 (1979). MAcHE, R., JALLIFFIER-VERNE, M., ROZIER, C., and LOISEAUX, S.: Molecular weight determination of precursor, mature and post-mature plastid ribosomal RNA from spinach using fully denaturing conditions. Biochim. Biophys. Acta 017,390-399 (1978).
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MUNSCHE, D., and WOLLGIEHN, R.: Die Synthese von ribosomaler RNA in Chloroplasten von Nicotiana rustica. Biochim. Biophys. Acta 294, 106-117 (1973). MUNSCHE, D., and WOLLGIEHN, R.: Altersabhiingige Labilitat der ribosomalen RNA aus Chloroplasten von Nicotiana rustica. Biochim. Biophys. Acta 340, 437-445 (1974). ROSNER, A., PORATH, D., and GRESSEL, J.: The distribution of plastid ribosomes and the integrity of plastid ribosomal RNA during the greening and maturation of Spirodela fronds. Plant and Cell Physiol. Hi, 891-902 (1974). SPEIRS, J., and GRIERSON, D.: Isolation and characterisation of 14-S RNA from spinach chloroplasts. Biochim. Biophys. Acta 1)21, 619-633 (1978). WRESCHNER, D., MELLOUL, D., and HERZBERG, M.: Interaction between membrane functions and protein synthesis in reticulocytes: Specific Cleavage of 28-S ribosomal RNA by a membrane constituent. Eur. J. Biochem. 81), 233-240 (1978). WOLLGIEHN, R., LERBS, S., and MUNSCHE, D.: Synthesis of ribosomal RNA in chloroplasts from tobacco leaves of different age. Biochem. Physiol. Pflanzen 170, 381-387 (1976). WOLLGIEHN, R., LERBS, S., and MUNSCHE, D.: Eigenschaften einer Chloroplasten-RNA vom Molekulargewicht 0.5 x 106 aus Nicotiana rustica. Bioehem. Physiol. Pflanzen 173, 60-69 (1978).
Received December 29, 1981 Authors' address: Dr. SILVA LERBS and Dr. REINHOLD WOLLGIEHN, Akademie der Wissenschaften der DDR, Forschungszentrum fUr Molekularbiologie und Medizin, Institut fur Biochemie der Pflanzen Halle (Saale), Weinberg 3, DDR - 4020 Halle (Saale).