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Immunological identification of a ribosomal protein on a sub-ribosomal particle
J. .MoZ. Biol. (1969) 41, 305-308
Immunological
Identification of a Ribosomal Protein on a Sub-ribosomal Particle
Bacteria grown under conditions which selectively inhibit protein synthesis fail to synthesize ribosomes (see review by Osawa, 1965). However, two ribonucleoprotein particles analogous to the 50 s and 30 s ribosomal subunits are formed. These particles, referred to here as sub-ribosomal particles, contain about 20 to 30% less protein than the corresponding subunits of mature ribosomes and are characterized by sedimentation coefficients of 23 to 25 s and 18 to 22 s (Kurland, Nomura & Watson, 1962; Dagley, Turnock & Wild, 1963). Physical and chemical studies demonstrate that the RNA isolated from these particles is very similar to the RNA isolated from mature ribosomal subunits (Kurland et al., 1962; Neidhardt & Eidlic, 1963; Dagley et al., 1963) and that similarly proteins isolated from these particles are ribosomal (Nakada, 1967). When the inhibition is removed, allowing the bacteria to resume protein synthesis, the sub-ribosomal particles disappear and concomitantly ribosomes appear. The particle RNA is now found in the newly synthesized ribosomes (Nakada, Anderson & Magasanik, 1964; Aronson BESpiegelman, 1961). The view that sub-ribosomal particles are precursors of mature ribosomes has been contested by Yoshida t Osawa (1968) and Schleif (1968), who suggested that sub-ribosomal particles, obtained from cells exposed to chloramphenicol, are non-specific aggregates of ribosomal RNA and protein. We report here on the reactivity of sub-ribosomal particles with an immune serum specific for a ribosomal protein. This serum was isolated from rabbits immunized with a suspension of 50 s “split proteins” from Escherichiu coli (Friedman, Olenick & Hahn, 1968). The split protein fraction was prepared by sedimentation of 50 s ribosomal subunits in cesium chloride, a procedure which removes 20% of the ribosomal proteins (split protein fraction), leaving ribonuolease-sensitive “core particles” (Me&son, Nomura, Brenner, Davern & Schlessinger, 1964). The immune serum reacts with both 50 s subunits and 70 s ribosomes, but does not react with 30 s subunits or core particles derived from either the 60 or 30 s subunit. Prebinding of transfer RNA to 70 s ribosomes or 50 s subunits inhibits the immune reaction, suggesting that the protein determinant studied is near, or perhaps is, the 50 s transfer RNA binding site (Friedman et aZ., 1968). Two types of sub-ribosomal particles, chloramphenicol particles and relaxed control particles, were studied. Both types of particles were isolated from E. c&i strainAB280, a methionine-requiring auxotroph relaxed in its control of RNA synthesis (RCrel ; Stent & BrennerJ961). Vigorously aerated cells were grown to a cell density of 108/ml. in M9 medium (Nakada et al., 1964) supplemented with 50 pg of nn-methionine/ml. Radioactively labeled chloramphenicol particles were isolated from cells exposed to 100 P-Qof chloramphenicol/ml. and [14C]uracil for 60 minutes. Radioactively labeled RC particles were isolated from washed cells which were incubated for 20 minutes in 306
306
D.
I.
FRIEDMAN,
A.
D.
WOLFE
AND
J. G. OLENICK
M9 medium without methionine and then an additional 100 minutes in the presence of [14C]uracil. In each case 10 pc of [14C]uracil (specific activity 1.3 po/pmole) were added to 200 ml. of bacterial culture. Cells were harvested in the cold, washed twice with 0.02 M-Tris-HCl, pH 74, containing O-002 M-magnesium acetate, and disrupted in a French pressure cell. The cell debris and unbroken cells were removed by centrifugation and the supernatant fluid was used in the following experiments. Radioactive ribosomal subunits, for use as marker, were isolated from cells exposed to [3H]uridine (final concentration of 0.1 PO/ml. with a specific activity of 2.6 PC/mole) during the final hour of growth.
400
I.5
350 300 250 200 150 100 50 1
IO
20 Fraction
30
40
no.
FIQ. 1. Sucrose gradient analysis of an extract containing chlorsmpheniool particles incubated with anti-60 s split protein serum. An extra& of 14C-labeled chloramphenicol partiales was prepared (see text). Ribosomal subunits (3H-labeled) were added and the 0.D .sao was adjusted to 20 units/ml. by adding unlabeled ribosomal subunits. Samples of the ilnal suspension were reacted for 20 min at 30% with equal volumes of either apeci6o antiserum or normal rabbit serum (each diluted 1 :lO in 0.02 M-Tris-HCl, pH 7.4, containing O-002 M-magnesium acetate). Samples (0.2 ml.) of each reaction mixture were layered on sucrose gradients (6 to 20% in 0.02 MTris-HCl, pH 7.4, containing 0.002 ~-magnesium acetate). The gradients were centrifuged at 100,000 g for 6 hr and collected in tubes oontaining 100 pg of carrier RNA. The fraotions were precipitated with cold 6% trichloroacetic acid. The precipitates were collected on membrane filters, suspended in a dioxane base scintillation fluid, and counted using a Paohard Tricarb liquid-scintillation speotrometer. Upper Fig. I control mixture reacted with normal rabbit serum; lower Fig. : mixture reacted with specific antiserum. (---) i*C; (-) sH. The arrows indicate (from left to right) 30s ribosomal subunits, large chloramphenicol particles, and small chloramphenicol particles.
LETTERS
TO
THE
307
EDITOR
00
50
00
5o
2
1
IO
20
h g :: "
30
100 .g
150
loo
50 1 1
I
I IO
1 20
Fraction
30 no.
FIQ. 2. Sucrose gradient analysis of an extract containing RC particles incubated v&h anti-SO s split protein *erum. An extract of ‘*C-labeled RC particles was prepared and analyzed as outlined in the legend to Fig. 1. Upper Fig. : control mixture reacted with normal rabbit serum ; lower Fig. : mixture reacted with sH. The arrows indicate (from left to right), 30s ribospecific antiserum. (----) W; (-) some1 subunits, large RC particles, and small RC particles.
The chloramphenicol and RC particles sediment in a sucrose gradient slower then the 30s subunit and faster than the soluble RNA peak (Figs 1 and 2, top). The bimodal sedimentation pattern suggests the presence of the two sub-ribosomal components demonstrated by Kurland et al. (1962) and Dagley et al. (1963). Normal rabbit serum does not affect the sedimentation behavior. Incubation with antiserum before sedimentation (Figs 1 and 2, bottom) significantly reduces only the faster sedimenting component of both the chloramphenicol and RC peaks. These experiments demonstrate that an antiserum specific for the split protein fraction isolated from 50 s ribosomal subunits reacts with the larger sub-ribosomal particle, but not with the smaller particle. Therefore, in both the case of ribosomes and sub-ribosomal particles, the split protein studied has a specific affinity for 23 s rRNA. This suggests that the formation of sub-ribosomal particles is not due to a random association of ribosomal RNA and protein into non-specific aggregates. A comparison between the 50 s ribosomal subunit and the larger sub-ribosomal particle has revealed a similrtrity in the RNA components. We show here that there is also a similarity with respect to a specific protein component.
308
D.
I.
FRIEDMAN,
A.
D.
WOLFE
AND
J.
G. OLENICK
We thank Dr F. E. Hahn for helpful discussion, encouragement and support. One of us (D. I. F.) is a post-doctoral fellow of the National Institute of General Medical Sciences, Fellowship no. l-F3-6M-23,762-01. Laboratory of Molecular Biology National Institute of Arthritis and Metabolic National Institutes of Health Bethesda, Md. 20014, U.S.A.
DAVID
Department of Molecular Biology Division of Medicine Walter Reed Army Institute of Research Washington, D.C. 20012, U.S.A. Received
1 November
I. FRIEDMAN
Diseases
1968, and in revised form 20 December
ALAN DAVID WOLFE JOHN G. OLENICK
1968
REFERENCES Aronson, A. I. $ Spiegelman, S. (1961). Biochim. 6io@ya. Acta, 53, 84. Dagley, S., Turnock, G. & Wild, D. G. (1963). Biochm. J. 88, 555. Friedman, D. I., Olenick, J. G. & Hahn, F. E. (1968). J. Mol. Biol. 32, 579. Kurland, C. G., Nomura, M. & Watson, J. D. (1962). J. Mol. Bid. 4, 388. Meselson, M., Nomura, M., Brenner, S., Davern, C. & Schlessinger, D. (1964). J. Mol. BioZ. 9, 696. Nakada, D. (1967). J. Mol. BioZ. 29, 473. Nakada, D., Anderson, I. A. & Magasanik, B. (1964). J. Mol. BioZ. 9, 472. Neidhart, F. C. & Eidlic, L. (1963). B&hint. biophys. Acta, 68, 380. Osawa, S. (1965). Prog. NucZeic Acid Res. 4, 161. Sohleif, R. F. (1968). J. Mol. BioZ. 37, 119. Stent, G. D. BEBrenner, S. (1961). Proc. Nat. Ad. Sk., Wash. 47, 2006. Yoshida, K. & Osawa, S. (1968). J. Mol. BioZ. 38, 659.
Immunological
Identification of a Ribosomal Protein on a Sub-ribosomal Particle
Bacteria grown under conditions which selectively inhibit protein synthesis fail to synthesize ribosomes (see review by Osawa, 1965). However, two ribonucleoprotein particles analogous to the 50 s and 30 s ribosomal subunits are formed. These particles, referred to here as sub-ribosomal particles, contain about 20 to 30% less protein than the corresponding subunits of mature ribosomes and are characterized by sedimentation coefficients of 23 to 25 s and 18 to 22 s (Kurland, Nomura & Watson, 1962; Dagley, Turnock & Wild, 1963). Physical and chemical studies demonstrate that the RNA isolated from these particles is very similar to the RNA isolated from mature ribosomal subunits (Kurland et al., 1962; Neidhardt & Eidlic, 1963; Dagley et al., 1963) and that similarly proteins isolated from these particles are ribosomal (Nakada, 1967). When the inhibition is removed, allowing the bacteria to resume protein synthesis, the sub-ribosomal particles disappear and concomitantly ribosomes appear. The particle RNA is now found in the newly synthesized ribosomes (Nakada, Anderson & Magasanik, 1964; Aronson BESpiegelman, 1961). The view that sub-ribosomal particles are precursors of mature ribosomes has been contested by Yoshida t Osawa (1968) and Schleif (1968), who suggested that sub-ribosomal particles, obtained from cells exposed to chloramphenicol, are non-specific aggregates of ribosomal RNA and protein. We report here on the reactivity of sub-ribosomal particles with an immune serum specific for a ribosomal protein. This serum was isolated from rabbits immunized with a suspension of 50 s “split proteins” from Escherichiu coli (Friedman, Olenick & Hahn, 1968). The split protein fraction was prepared by sedimentation of 50 s ribosomal subunits in cesium chloride, a procedure which removes 20% of the ribosomal proteins (split protein fraction), leaving ribonuolease-sensitive “core particles” (Me&son, Nomura, Brenner, Davern & Schlessinger, 1964). The immune serum reacts with both 50 s subunits and 70 s ribosomes, but does not react with 30 s subunits or core particles derived from either the 60 or 30 s subunit. Prebinding of transfer RNA to 70 s ribosomes or 50 s subunits inhibits the immune reaction, suggesting that the protein determinant studied is near, or perhaps is, the 50 s transfer RNA binding site (Friedman et aZ., 1968). Two types of sub-ribosomal particles, chloramphenicol particles and relaxed control particles, were studied. Both types of particles were isolated from E. c&i strainAB280, a methionine-requiring auxotroph relaxed in its control of RNA synthesis (RCrel ; Stent & BrennerJ961). Vigorously aerated cells were grown to a cell density of 108/ml. in M9 medium (Nakada et al., 1964) supplemented with 50 pg of nn-methionine/ml. Radioactively labeled chloramphenicol particles were isolated from cells exposed to 100 P-Qof chloramphenicol/ml. and [14C]uracil for 60 minutes. Radioactively labeled RC particles were isolated from washed cells which were incubated for 20 minutes in 306
306
D.
I.
FRIEDMAN,
A.
D.
WOLFE
AND
J. G. OLENICK
M9 medium without methionine and then an additional 100 minutes in the presence of [14C]uracil. In each case 10 pc of [14C]uracil (specific activity 1.3 po/pmole) were added to 200 ml. of bacterial culture. Cells were harvested in the cold, washed twice with 0.02 M-Tris-HCl, pH 74, containing O-002 M-magnesium acetate, and disrupted in a French pressure cell. The cell debris and unbroken cells were removed by centrifugation and the supernatant fluid was used in the following experiments. Radioactive ribosomal subunits, for use as marker, were isolated from cells exposed to [3H]uridine (final concentration of 0.1 PO/ml. with a specific activity of 2.6 PC/mole) during the final hour of growth.
400
I.5
350 300 250 200 150 100 50 1
IO
20 Fraction
30
40
no.
FIQ. 1. Sucrose gradient analysis of an extract containing chlorsmpheniool particles incubated with anti-60 s split protein serum. An extra& of 14C-labeled chloramphenicol partiales was prepared (see text). Ribosomal subunits (3H-labeled) were added and the 0.D .sao was adjusted to 20 units/ml. by adding unlabeled ribosomal subunits. Samples of the ilnal suspension were reacted for 20 min at 30% with equal volumes of either apeci6o antiserum or normal rabbit serum (each diluted 1 :lO in 0.02 M-Tris-HCl, pH 7.4, containing O-002 M-magnesium acetate). Samples (0.2 ml.) of each reaction mixture were layered on sucrose gradients (6 to 20% in 0.02 MTris-HCl, pH 7.4, containing 0.002 ~-magnesium acetate). The gradients were centrifuged at 100,000 g for 6 hr and collected in tubes oontaining 100 pg of carrier RNA. The fraotions were precipitated with cold 6% trichloroacetic acid. The precipitates were collected on membrane filters, suspended in a dioxane base scintillation fluid, and counted using a Paohard Tricarb liquid-scintillation speotrometer. Upper Fig. I control mixture reacted with normal rabbit serum; lower Fig. : mixture reacted with specific antiserum. (---) i*C; (-) sH. The arrows indicate (from left to right) 30s ribosomal subunits, large chloramphenicol particles, and small chloramphenicol particles.
LETTERS
TO
THE
307
EDITOR
00
50
00
5o
2
1
IO
20
h g :: "
30
100 .g
150
loo
50 1 1
I
I IO
1 20
Fraction
30 no.
FIQ. 2. Sucrose gradient analysis of an extract containing RC particles incubated v&h anti-SO s split protein *erum. An extract of ‘*C-labeled RC particles was prepared and analyzed as outlined in the legend to Fig. 1. Upper Fig. : control mixture reacted with normal rabbit serum ; lower Fig. : mixture reacted with sH. The arrows indicate (from left to right), 30s ribospecific antiserum. (----) W; (-) some1 subunits, large RC particles, and small RC particles.
The chloramphenicol and RC particles sediment in a sucrose gradient slower then the 30s subunit and faster than the soluble RNA peak (Figs 1 and 2, top). The bimodal sedimentation pattern suggests the presence of the two sub-ribosomal components demonstrated by Kurland et al. (1962) and Dagley et al. (1963). Normal rabbit serum does not affect the sedimentation behavior. Incubation with antiserum before sedimentation (Figs 1 and 2, bottom) significantly reduces only the faster sedimenting component of both the chloramphenicol and RC peaks. These experiments demonstrate that an antiserum specific for the split protein fraction isolated from 50 s ribosomal subunits reacts with the larger sub-ribosomal particle, but not with the smaller particle. Therefore, in both the case of ribosomes and sub-ribosomal particles, the split protein studied has a specific affinity for 23 s rRNA. This suggests that the formation of sub-ribosomal particles is not due to a random association of ribosomal RNA and protein into non-specific aggregates. A comparison between the 50 s ribosomal subunit and the larger sub-ribosomal particle has revealed a similrtrity in the RNA components. We show here that there is also a similarity with respect to a specific protein component.
308
D.
I.
FRIEDMAN,
A.
D.
WOLFE
AND
J.
G. OLENICK
We thank Dr F. E. Hahn for helpful discussion, encouragement and support. One of us (D. I. F.) is a post-doctoral fellow of the National Institute of General Medical Sciences, Fellowship no. l-F3-6M-23,762-01. Laboratory of Molecular Biology National Institute of Arthritis and Metabolic National Institutes of Health Bethesda, Md. 20014, U.S.A.
DAVID
Department of Molecular Biology Division of Medicine Walter Reed Army Institute of Research Washington, D.C. 20012, U.S.A. Received
1 November
I. FRIEDMAN
Diseases
1968, and in revised form 20 December
ALAN DAVID WOLFE JOHN G. OLENICK
1968
REFERENCES Aronson, A. I. $ Spiegelman, S. (1961). Biochim. 6io@ya. Acta, 53, 84. Dagley, S., Turnock, G. & Wild, D. G. (1963). Biochm. J. 88, 555. Friedman, D. I., Olenick, J. G. & Hahn, F. E. (1968). J. Mol. Biol. 32, 579. Kurland, C. G., Nomura, M. & Watson, J. D. (1962). J. Mol. Bid. 4, 388. Meselson, M., Nomura, M., Brenner, S., Davern, C. & Schlessinger, D. (1964). J. Mol. BioZ. 9, 696. Nakada, D. (1967). J. Mol. BioZ. 29, 473. Nakada, D., Anderson, I. A. & Magasanik, B. (1964). J. Mol. BioZ. 9, 472. Neidhart, F. C. & Eidlic, L. (1963). B&hint. biophys. Acta, 68, 380. Osawa, S. (1965). Prog. NucZeic Acid Res. 4, 161. Sohleif, R. F. (1968). J. Mol. BioZ. 37, 119. Stent, G. D. BEBrenner, S. (1961). Proc. Nat. Ad. Sk., Wash. 47, 2006. Yoshida, K. & Osawa, S. (1968). J. Mol. BioZ. 38, 659.