Proc. NatL. Acad. Sci. USA Vol. 73, No. 5, pp. 1547-1551, May 1976
Biochemistry
Coordinate regulation of the synthesis of eukaryotic ribosomal proteins (mRNA/yeast/temperature sensitive mutants)
CHARLES GORENSTEIN AND JONATHAN R. WARNER Departments of Biochemistry and Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461
Communicated by Harry Eagle, February 26, 1976
ABSTRACT We have developed a method for the direct measurement, in eukaryotic cells, of the synthesis of ribosomal proteins, irrespective of the synthesis of ribosomes. In this way the synthesis of ribosomal proteins has been examined in mutant strains of Saccharomyces cerevisiae, which are unable to synthesize ribosomes under nonpermissive conditions. The results suggest that the synthesis of more than 40 ribosomal proteins is under coordinate control. Under nonpermissive conditions, the synthesis of each protein declines exponentially to a basal level which is 10-20% of normal. The kinetics of that decline suggest that an early, if not primary, result of the nonpermissive conditions is the cessation of production of new mRNA for-each of the ribosomal proteins. The coordinate regulation appears not to be influenced directly by the rate of transcription of ribosomal precursor RNA. The eukaryotic ribosome is assembled from several species of RNA, synthesized within the nucleus, and more than 50 proteins, synthesized in the cytoplasm (1). The genetically tractable eukaryote, Saccharomyces cerevisiae, is useful for studying the coordination between such nuclear and cytoplasmic processes. Hartwell and coworkers have identified 10 unlinked genes, rna 2-rna 11, in which temperature sensitive mutants are unable to synthesize ribosomes at the restrictive temperature (2), although protein synthesis continues normally. While such cells continue to transcribe ribosomal precursor RNA at up to 80% of the control rate (ref. 3; R. Shulman, unpublished), the RNA is not properly processed, and is eventually degraded. Indirect experiments suggested that the synthesis of ribosomal proteins is also inhibited at the restrictive temperature (3). In the present communication we describe a method, adapted from that of Dennis (4), to measure the synthesis of ribosomal proteins directly. We show that mutations in gene ma 2 or gene rna 9 cause the coordinate inhibition of the synthesis of at least 40 ribosomal proteins. The kinetics of the inhibition suggest that an early effect of each mutation is the suppression of the synthesis of mRNA for the ribosomal proteins.
METHODS Cells and Media. The parent strain of S. cerevisiae, A364A, (ATCC no. 22244) was obtained from L. Hartwell. Temperature sensitive mutants derived from this strain are: ts 136 (rna 1-), in which no RNA passes from the nucleus to the cytoplasm at the restrictive temperature, 36° (5); ts 368 (rna 2-), and ts 257 (ma 9-), in which no ribosomes are made at temperatures of 330 or above (2). Media, growth conditions, and procedures for preparing spheroplasts have been described (6, 7). Analysis of Data. The experiments described below involve cells labeled uniformly with ["4C]leucine for several generations and pulsed for a short period with [3H]leucine as described in the legend to Fig. 2. The 14C values for individual proteins are Abbreviations: Bistris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1,3-propanediol; ts, temperature sensitive. 1547
reproducible to within 10%, indicating not only that the yield during extraction is consistent, but also that the [14C]protein is stable. Under these conditions the 3H/14C ratio of any protein is a measure of the synthesis of that protein at the time of the pulse. When the culture is shifted to the elevated temperature, there is an instantaneous doubling of the rate of incorporation of amino acids. Therefore, to compare the rate of ribosomal protein synthesis at the elevated temperature to that at 230, we expressed the relative rate of synthesis of each protein as Ai (4), where Ai =
(3H/14C in ith protein) (3H/"4C in total cell protein)'
In this form, if Ai = 1, the protein was synthesized, in relation to its abundance in the cell, at the same rate as the total cell protein, if Ai < 1, the corresponding rate was less than that of the total cell protein. Since only the 3H/14C ratio of any protein is used as data, the yield of the protein after analysis is not critical. Analysis of Ribosomal Proteins. To study the synthesis of ribosomal proteins directly, we extracted the total protein complement of the cell and analyzed it by two-dimensional polyacrylamide gel electrophoresis. Because most of the ribosomal proteins are small and basic, they can be clearly separated from nonribosomal proteins (Fig. 1). The ribosomal proteins in Fig. lb have been designated by numbers and identified with an individual subunit (Table 1). We have detected no differences between the ribosomal proteins of the wild type and those of any mutant used in this study. Some nonribosomal proteins of Fig. la have been designated by letters for use as controls. That ribosomal proteins, whether part of a ribosome or not, are extracted with high efficiency is demonstrated by the finding that their Ai is approximately 1 after a five-min pulse, although newly synthesized ribosomal proteins are found on ribosomes only after a 10-min lag (7). Their Ai does not increase after a chase with unlabeled amino acid.
RESULTS of Synthesis Ribosomal Proteins at the Restrictive Temperature. We wish to ask whether the synthesis of ribosomal proteins continues at the restrictive temperature, in mutants which can no longer synthesize ribosomes. The temperature sensitivity of the mutants is not instantaneous but requires 30-60 min for full expression (3). Therefore, in the first experiment we have measured the synthesis of ribosomal proteins at the permissive temperature, as well as after 90 min at the restrictive temperature, using the wild type, rna+, and two mutants, with lesions in genes ma 2 and rna 9. The data for three ribosomal proteins are presented in Fig. 2. In the mutants there has been a substantial inhibition of synthesis of ribosomal proteins in spite of the continued synthesis of the bulk of the cells' proteins (ref.
1548
]Biochemistry: Gorenstein and Warner pH
Proc. Natl. Acad. Sci. USA 73 (1976)
5
sodium dodecyl sulfate
A
A.
,
li s
..*
a
i-> pH 5 sodium dodecyl sulfate
_W2 3
5 _8 w~
14
13
12
409 4,
11
15
21*6 40 *39 * 2
4I> O
2
i1 *5
3
65
29
* 48
46
63
b FIG. 1. Two-dimensional gel electropherograms of yeast total protein (a) and ribosomal protein (b). (a) A culture containing 4 X 107 spheroplasts was poured over crushed, frozen 1 M sorbitol, containing cycloheximide at 100 jsg/ml final concentration, and the cells were collected by centrifugation at 10,000 X g for 5 min. The cells were lysed in 1 ml of distilled water, followed immediately by the addition of 0.1 volume of 1 M MgCl2 and two volumes of glacial acetic acid. The cell lysate was stirred for 30 min in the cold; ribosomal RNA was removed by centrifugation at 20,000 X g for 15 min. The supernatant, containing 95% of the cells' protein, was dialyzed against 1% acetic acid, lyophilized, dissolved in 100 Ml of sample buffer (0.1% acetic acid, 8 M urea, 1.4 M mercaptoethanol, 10% glycerol), and subjected to two-dimensional gel electrophoresis. The gel for the first dimension was formed in a glass cylinder 12 X 0.5 cm, and consisted of 4% acrylamide, 0.1% bisacrylamide, 8 M urea, 0.057 M 2-[bis(2-hydroxyethyl)amino]-2(hydroxymethyl)-1,3-propanediol (Bistris), adjusted to pH 5 with acetic acid (12). The upper buffer was 0.01 M Bistris adjusted to pH 4 with acetic acid. The lower buffer was 0.179 M potassium acetate, pH 5. Electrophoresis towards the cathode was at 150 V for 10 hr. The gel was removed from its tube, equilibrated for 45 min in 1% sodium dodecyl sulfate, 0.5 M Tris, pH 6.8, and sealed in the top of a two-dimensional gel apparatus (15 X 10 X 0.3 cm). The second dimension consisted of a 17% acrylamide resolving gel, pH 8.6, overlaid with a 4% stacking gel, pH 6.8 (13). Electrophoresis was for 12 hr at 150 V. The gels were stained for 5 hr with 0.2% Coomassie blue in 50% methanol, and destained in 30% methanol for several days. (b) Ribosomes were prepared as described (14). Their proteins were extracted, and 200 Mg were subjected to electrophoresis as described above.
2; Fig. 4 insets). Table 1 demonstrates that, with a few exceptions, the synthesis of each of the ribosomal proteins responds in parallel to the restrictive temperature. Experiments using mutants in genes rna 4, rna 5, rna 6, or rna 10 gave similar results, both for those proteins whose synthesis is coordinately repressed and for those proteins whose
synthesis continues. Decreased Synthesis or Increased Degradation. In certain
experimental situations in mammalian cells, ribosomal proteins that are not immediately used may be subsequently degraded (1, 8). In order to determine if the data described above were due to lack of synthesis of ribosomal proteins, or to normal synthesis followed by decay, we carried out the experiment described in Fig. 3. In these cells the accumulation of ribosomal [3H]proteins, although reduced by 90% or more at 330, appears to be linear, and the labeled proteins are stable during the chase.
Gorenstein and Warner
Biochemistry:
Proc. Natl. Acad. Sci. USA 73 (1976)
Table 1. Synthesis of ribosomal and nonribosomal proteins at permissive and restrictive temperatures
A364A No.
Subunit
ts 368
23° 1.0
ts 257
230
330
230
330
0.77 0.79 1.05 0.88 0.76 2.67 0.93 0.84 0.90 1.20 0.83 0.83 0.78 0.78 0.83 0.84 0.66 0.95 0.73 0.66 0.82 0.85 0.85 0.95 0.81 1.06 0.86 0.96 0.95 0.93 0.93 0.76 0.70 0.85 0.75 0.97 0.95 0.89 0.84 0.89 ? 54 0.81 0.85 S 55 0.84 0.85 59 0.80 0.82 I? 61 0.89 0.79 62 L 0.74 0.82 63 S 0.76 0.70 64 L 0.77 0.70 65 L 0.70 0.67 Nonribosomal proteins A 0.77 1.10 B 0.95 0.83 C 0.94 1.06
0.90 1.01 1.07 1.33 1.08 3.54 1.0 1.10 1.24 1.54 0.91 1.33 1.08 1.19 1.15 0.92 1.03 1.22 1.15 1.15 1.12 1.21 1.15 1.19 1.08 1.03 1.07 1.08 1.22 1.08 1.20 0.96 0.86 0.86 1.03 1.24 0.96 1.07 0.96 1.14 1.15 0.98 1.10 0.98 0.88 0.94 0.98 0.94
0.31 0.40 1.56 0.09 0.10 0.95 0.12 0.50 0.57 1.23 0.25 1.02 0.10 0.11 0.12 0.08 0.08 0.18 1.06 1.06 0.16 0.05 0.11 0.21 0.14 0.23 0.10 0.17 0.20 1.63 0.22 0.18 0.19 0.15 0.10 0.14 0.16 0.15 0.23 0.18 0.16 0.16 0.23 0.17 0.15 0.12 0.13 0.06
0.80 0.85 1.10 0.94 0.96 3.41 0.93 0.93 0.95 1.15 0.90 0.92 0.91 0.98 0.99 0.92 0.98 0.98 0.84 0.95 0.94 0.94 0.96 1.02 0.90 0.94 0.93 1.04 1.0 0.90 1.02 0.99 0.97 0.98 1.0 1.04 0.98 1.20 1.08 0.95 1.17 1.10 1.06 1.06 1.04 0.96 0.95 1.03
0.24 0.34 1.05 0.12 0.14 1.51 0.15 0.35 0.40 1.15 0.18 0.49 0.15 0.14 0.14 0.11 0.09 0.21 0.70 0.70 0.19 0.06 0.14 0.41 0.20 0.36 0.11 0.20 0.23 1.07 0.23 0.27 0.19 0.19 0.15 0.16 0.16 0.18 0.20 0.19 0.26 0.22 0.31 0.24 0.19 0.12 0.18 0.10
0.87 1.07 1.09
1.09 1.17 1.26
0.89 0.90 1.28
0.98 1.0 1.44
Ribosomal proteins 1 0.85 L 0.89 2 L 0.93 L 3* S 1.01 5 L 0.89 8 3.03 9 S 11 1.13 L S 12 1.0 S 13 0.90 S 14* 1.16 L 15 0.82 L 16* 0.89 L 18 0.82 S 19 0.79 S 0.70 20 21 S 0.93 22 L 0.79 23 L 0.95 24* L 0.82 L 25* 0.86 L 27 0.83 28 L 0.87 L 29 0.77 S 30 1.09 31 L 0.81 32 L 0.73 33 L 0.83 S 37 0.84 L 38 0.84 39* L 0.92 S 40 0.79 41 S 0.74 44 L 0.60 45 L 0.75 9 46 0.76 L 47 0.77 L 48 0.80 S 50 0.89 52 S 0.90 9 53 0.79
Orna +
rna
330
230
330
1549
2
E-r
0.5
The rate of synthesis (Ai) of ribosomal proteins and some nonribosomal proteins was determined at 230, and after 90 min at 330, as described in the legend of Fig. 2. See Fig. 1 for the number scheme of the proteins. The subunit designation of each protein was derived from gels of proteins prepared from 60S (L) and 40S (S) subunits isolated in buffer containing 0.5M KCl. Asterisks indicate ribosomal proteins that do not appear to be under coordinate control.
8
21 1 PROTEIN NUMBER
28
FIG. 2. Synthesis of three ribosomal proteins at the permissive and the restrictive temperatures. Cultures of A364A, ts 368, and ts 257 were grown at 230 in synthetic medium containing [14C]leucine (2.5 ,Ci/ml) for five to six generations. They were washed, converted to spheroplasts, and resuspended in synthetic medium containing 0.4 M MgSO4 for osmotic support. After 2 hr a portion of each culture was shifted to the restrictive temperature, 33°. After 90 min more each of the six cultures was labeled with [3H]leucine (100 MCi/ml) for 5 min and harvested, and the total protein was prepared as described in the legend to Fig. 1. The protein was mixed with marker ribosomal protein and electrophoresed as described in Fig. 1. Spots corresponding to ribosomal and nonribosomal proteins were excised with a cork borer and treated at 600 in capped glass vials with 0.5 ml of 30% H202. Radioactivity was determined in 10 ml of Aquasol (New England Nuclear Corp.). The relative synthetic rate of each protein, Ai, was calculated as described in the text. This is a sample of the data presented in Table 1.
We conclude that the synthesis of the ribosomal proteins is coordinately repressed at the restrictive temperature. Kinetics of Inhibition of Ribosomal Protein Synthesis. To provide some insight into the mechanism by which the coordinate control of ribosomal protein synthesis occurs, we have studied the kinetics of the inhibition of ribosomal protein synthesis when a mutant is shifted from the permissive to the restrictive temperature (Fig. 4). In the wild type, although the nonribosomal proteins continue to be made at a relatively normal rate, there is a transient inhibition of the synthesis of ribosomal proteins (Fig. 4a). This finding is consistent with earlier reports of a transient inhibition of ribosome synthesis after a temperature increase (3). In cells carrying the mutation in rna 2, the synthesis of nonribosomal proteins continues normally, whereas the syn-
C.
O
!.0 '1,
z
z
PO,
90
DI 40 60 TIME (MINUTES)
FIG. 3. Stability of ribosomal proteins synthesized at the permissive and restrictive temperatures. A culture of ts 368 was grown overnight in [14C]leucine; half of the culture was kept at 230, the other half was shifted to 330 for 60 min. Spheroplasts were then pulsed for 20 min with [3H]leucine and chased for 60 min with unlabeled leucine (1 mg/ml). Aliquots were removed during the pulse and the chase, Ribosomal protein synthesis was followed as described in Fig. 2. The data are presented as the 3H/14C ratio for ribosomal proteins no. 8 (0) and no. 18 (@).(--- -) 230; (- ) 330. Similar data were obtained for thirty of the proteins listed in Table 1.
1550
Biochemistry: Gorenstein and Warner
Proc. Nati. Acad. Sci. USA 73 (1976)
I_
Biochemistry
Coordinate regulation of the synthesis of eukaryotic ribosomal proteins (mRNA/yeast/temperature sensitive mutants)
CHARLES GORENSTEIN AND JONATHAN R. WARNER Departments of Biochemistry and Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461
Communicated by Harry Eagle, February 26, 1976
ABSTRACT We have developed a method for the direct measurement, in eukaryotic cells, of the synthesis of ribosomal proteins, irrespective of the synthesis of ribosomes. In this way the synthesis of ribosomal proteins has been examined in mutant strains of Saccharomyces cerevisiae, which are unable to synthesize ribosomes under nonpermissive conditions. The results suggest that the synthesis of more than 40 ribosomal proteins is under coordinate control. Under nonpermissive conditions, the synthesis of each protein declines exponentially to a basal level which is 10-20% of normal. The kinetics of that decline suggest that an early, if not primary, result of the nonpermissive conditions is the cessation of production of new mRNA for-each of the ribosomal proteins. The coordinate regulation appears not to be influenced directly by the rate of transcription of ribosomal precursor RNA. The eukaryotic ribosome is assembled from several species of RNA, synthesized within the nucleus, and more than 50 proteins, synthesized in the cytoplasm (1). The genetically tractable eukaryote, Saccharomyces cerevisiae, is useful for studying the coordination between such nuclear and cytoplasmic processes. Hartwell and coworkers have identified 10 unlinked genes, rna 2-rna 11, in which temperature sensitive mutants are unable to synthesize ribosomes at the restrictive temperature (2), although protein synthesis continues normally. While such cells continue to transcribe ribosomal precursor RNA at up to 80% of the control rate (ref. 3; R. Shulman, unpublished), the RNA is not properly processed, and is eventually degraded. Indirect experiments suggested that the synthesis of ribosomal proteins is also inhibited at the restrictive temperature (3). In the present communication we describe a method, adapted from that of Dennis (4), to measure the synthesis of ribosomal proteins directly. We show that mutations in gene ma 2 or gene rna 9 cause the coordinate inhibition of the synthesis of at least 40 ribosomal proteins. The kinetics of the inhibition suggest that an early effect of each mutation is the suppression of the synthesis of mRNA for the ribosomal proteins.
METHODS Cells and Media. The parent strain of S. cerevisiae, A364A, (ATCC no. 22244) was obtained from L. Hartwell. Temperature sensitive mutants derived from this strain are: ts 136 (rna 1-), in which no RNA passes from the nucleus to the cytoplasm at the restrictive temperature, 36° (5); ts 368 (rna 2-), and ts 257 (ma 9-), in which no ribosomes are made at temperatures of 330 or above (2). Media, growth conditions, and procedures for preparing spheroplasts have been described (6, 7). Analysis of Data. The experiments described below involve cells labeled uniformly with ["4C]leucine for several generations and pulsed for a short period with [3H]leucine as described in the legend to Fig. 2. The 14C values for individual proteins are Abbreviations: Bistris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1,3-propanediol; ts, temperature sensitive. 1547
reproducible to within 10%, indicating not only that the yield during extraction is consistent, but also that the [14C]protein is stable. Under these conditions the 3H/14C ratio of any protein is a measure of the synthesis of that protein at the time of the pulse. When the culture is shifted to the elevated temperature, there is an instantaneous doubling of the rate of incorporation of amino acids. Therefore, to compare the rate of ribosomal protein synthesis at the elevated temperature to that at 230, we expressed the relative rate of synthesis of each protein as Ai (4), where Ai =
(3H/14C in ith protein) (3H/"4C in total cell protein)'
In this form, if Ai = 1, the protein was synthesized, in relation to its abundance in the cell, at the same rate as the total cell protein, if Ai < 1, the corresponding rate was less than that of the total cell protein. Since only the 3H/14C ratio of any protein is used as data, the yield of the protein after analysis is not critical. Analysis of Ribosomal Proteins. To study the synthesis of ribosomal proteins directly, we extracted the total protein complement of the cell and analyzed it by two-dimensional polyacrylamide gel electrophoresis. Because most of the ribosomal proteins are small and basic, they can be clearly separated from nonribosomal proteins (Fig. 1). The ribosomal proteins in Fig. lb have been designated by numbers and identified with an individual subunit (Table 1). We have detected no differences between the ribosomal proteins of the wild type and those of any mutant used in this study. Some nonribosomal proteins of Fig. la have been designated by letters for use as controls. That ribosomal proteins, whether part of a ribosome or not, are extracted with high efficiency is demonstrated by the finding that their Ai is approximately 1 after a five-min pulse, although newly synthesized ribosomal proteins are found on ribosomes only after a 10-min lag (7). Their Ai does not increase after a chase with unlabeled amino acid.
RESULTS of Synthesis Ribosomal Proteins at the Restrictive Temperature. We wish to ask whether the synthesis of ribosomal proteins continues at the restrictive temperature, in mutants which can no longer synthesize ribosomes. The temperature sensitivity of the mutants is not instantaneous but requires 30-60 min for full expression (3). Therefore, in the first experiment we have measured the synthesis of ribosomal proteins at the permissive temperature, as well as after 90 min at the restrictive temperature, using the wild type, rna+, and two mutants, with lesions in genes ma 2 and rna 9. The data for three ribosomal proteins are presented in Fig. 2. In the mutants there has been a substantial inhibition of synthesis of ribosomal proteins in spite of the continued synthesis of the bulk of the cells' proteins (ref.
1548
]Biochemistry: Gorenstein and Warner pH
Proc. Natl. Acad. Sci. USA 73 (1976)
5
sodium dodecyl sulfate
A
A.
,
li s
..*
a
i-> pH 5 sodium dodecyl sulfate
_W2 3
5 _8 w~
14
13
12
409 4,
11
15
21*6 40 *39 * 2
4I> O
2
i1 *5
3
65
29
* 48
46
63
b FIG. 1. Two-dimensional gel electropherograms of yeast total protein (a) and ribosomal protein (b). (a) A culture containing 4 X 107 spheroplasts was poured over crushed, frozen 1 M sorbitol, containing cycloheximide at 100 jsg/ml final concentration, and the cells were collected by centrifugation at 10,000 X g for 5 min. The cells were lysed in 1 ml of distilled water, followed immediately by the addition of 0.1 volume of 1 M MgCl2 and two volumes of glacial acetic acid. The cell lysate was stirred for 30 min in the cold; ribosomal RNA was removed by centrifugation at 20,000 X g for 15 min. The supernatant, containing 95% of the cells' protein, was dialyzed against 1% acetic acid, lyophilized, dissolved in 100 Ml of sample buffer (0.1% acetic acid, 8 M urea, 1.4 M mercaptoethanol, 10% glycerol), and subjected to two-dimensional gel electrophoresis. The gel for the first dimension was formed in a glass cylinder 12 X 0.5 cm, and consisted of 4% acrylamide, 0.1% bisacrylamide, 8 M urea, 0.057 M 2-[bis(2-hydroxyethyl)amino]-2(hydroxymethyl)-1,3-propanediol (Bistris), adjusted to pH 5 with acetic acid (12). The upper buffer was 0.01 M Bistris adjusted to pH 4 with acetic acid. The lower buffer was 0.179 M potassium acetate, pH 5. Electrophoresis towards the cathode was at 150 V for 10 hr. The gel was removed from its tube, equilibrated for 45 min in 1% sodium dodecyl sulfate, 0.5 M Tris, pH 6.8, and sealed in the top of a two-dimensional gel apparatus (15 X 10 X 0.3 cm). The second dimension consisted of a 17% acrylamide resolving gel, pH 8.6, overlaid with a 4% stacking gel, pH 6.8 (13). Electrophoresis was for 12 hr at 150 V. The gels were stained for 5 hr with 0.2% Coomassie blue in 50% methanol, and destained in 30% methanol for several days. (b) Ribosomes were prepared as described (14). Their proteins were extracted, and 200 Mg were subjected to electrophoresis as described above.
2; Fig. 4 insets). Table 1 demonstrates that, with a few exceptions, the synthesis of each of the ribosomal proteins responds in parallel to the restrictive temperature. Experiments using mutants in genes rna 4, rna 5, rna 6, or rna 10 gave similar results, both for those proteins whose synthesis is coordinately repressed and for those proteins whose
synthesis continues. Decreased Synthesis or Increased Degradation. In certain
experimental situations in mammalian cells, ribosomal proteins that are not immediately used may be subsequently degraded (1, 8). In order to determine if the data described above were due to lack of synthesis of ribosomal proteins, or to normal synthesis followed by decay, we carried out the experiment described in Fig. 3. In these cells the accumulation of ribosomal [3H]proteins, although reduced by 90% or more at 330, appears to be linear, and the labeled proteins are stable during the chase.
Gorenstein and Warner
Biochemistry:
Proc. Natl. Acad. Sci. USA 73 (1976)
Table 1. Synthesis of ribosomal and nonribosomal proteins at permissive and restrictive temperatures
A364A No.
Subunit
ts 368
23° 1.0
ts 257
230
330
230
330
0.77 0.79 1.05 0.88 0.76 2.67 0.93 0.84 0.90 1.20 0.83 0.83 0.78 0.78 0.83 0.84 0.66 0.95 0.73 0.66 0.82 0.85 0.85 0.95 0.81 1.06 0.86 0.96 0.95 0.93 0.93 0.76 0.70 0.85 0.75 0.97 0.95 0.89 0.84 0.89 ? 54 0.81 0.85 S 55 0.84 0.85 59 0.80 0.82 I? 61 0.89 0.79 62 L 0.74 0.82 63 S 0.76 0.70 64 L 0.77 0.70 65 L 0.70 0.67 Nonribosomal proteins A 0.77 1.10 B 0.95 0.83 C 0.94 1.06
0.90 1.01 1.07 1.33 1.08 3.54 1.0 1.10 1.24 1.54 0.91 1.33 1.08 1.19 1.15 0.92 1.03 1.22 1.15 1.15 1.12 1.21 1.15 1.19 1.08 1.03 1.07 1.08 1.22 1.08 1.20 0.96 0.86 0.86 1.03 1.24 0.96 1.07 0.96 1.14 1.15 0.98 1.10 0.98 0.88 0.94 0.98 0.94
0.31 0.40 1.56 0.09 0.10 0.95 0.12 0.50 0.57 1.23 0.25 1.02 0.10 0.11 0.12 0.08 0.08 0.18 1.06 1.06 0.16 0.05 0.11 0.21 0.14 0.23 0.10 0.17 0.20 1.63 0.22 0.18 0.19 0.15 0.10 0.14 0.16 0.15 0.23 0.18 0.16 0.16 0.23 0.17 0.15 0.12 0.13 0.06
0.80 0.85 1.10 0.94 0.96 3.41 0.93 0.93 0.95 1.15 0.90 0.92 0.91 0.98 0.99 0.92 0.98 0.98 0.84 0.95 0.94 0.94 0.96 1.02 0.90 0.94 0.93 1.04 1.0 0.90 1.02 0.99 0.97 0.98 1.0 1.04 0.98 1.20 1.08 0.95 1.17 1.10 1.06 1.06 1.04 0.96 0.95 1.03
0.24 0.34 1.05 0.12 0.14 1.51 0.15 0.35 0.40 1.15 0.18 0.49 0.15 0.14 0.14 0.11 0.09 0.21 0.70 0.70 0.19 0.06 0.14 0.41 0.20 0.36 0.11 0.20 0.23 1.07 0.23 0.27 0.19 0.19 0.15 0.16 0.16 0.18 0.20 0.19 0.26 0.22 0.31 0.24 0.19 0.12 0.18 0.10
0.87 1.07 1.09
1.09 1.17 1.26
0.89 0.90 1.28
0.98 1.0 1.44
Ribosomal proteins 1 0.85 L 0.89 2 L 0.93 L 3* S 1.01 5 L 0.89 8 3.03 9 S 11 1.13 L S 12 1.0 S 13 0.90 S 14* 1.16 L 15 0.82 L 16* 0.89 L 18 0.82 S 19 0.79 S 0.70 20 21 S 0.93 22 L 0.79 23 L 0.95 24* L 0.82 L 25* 0.86 L 27 0.83 28 L 0.87 L 29 0.77 S 30 1.09 31 L 0.81 32 L 0.73 33 L 0.83 S 37 0.84 L 38 0.84 39* L 0.92 S 40 0.79 41 S 0.74 44 L 0.60 45 L 0.75 9 46 0.76 L 47 0.77 L 48 0.80 S 50 0.89 52 S 0.90 9 53 0.79
Orna +
rna
330
230
330
1549
2
E-r
0.5
The rate of synthesis (Ai) of ribosomal proteins and some nonribosomal proteins was determined at 230, and after 90 min at 330, as described in the legend of Fig. 2. See Fig. 1 for the number scheme of the proteins. The subunit designation of each protein was derived from gels of proteins prepared from 60S (L) and 40S (S) subunits isolated in buffer containing 0.5M KCl. Asterisks indicate ribosomal proteins that do not appear to be under coordinate control.
8
21 1 PROTEIN NUMBER
28
FIG. 2. Synthesis of three ribosomal proteins at the permissive and the restrictive temperatures. Cultures of A364A, ts 368, and ts 257 were grown at 230 in synthetic medium containing [14C]leucine (2.5 ,Ci/ml) for five to six generations. They were washed, converted to spheroplasts, and resuspended in synthetic medium containing 0.4 M MgSO4 for osmotic support. After 2 hr a portion of each culture was shifted to the restrictive temperature, 33°. After 90 min more each of the six cultures was labeled with [3H]leucine (100 MCi/ml) for 5 min and harvested, and the total protein was prepared as described in the legend to Fig. 1. The protein was mixed with marker ribosomal protein and electrophoresed as described in Fig. 1. Spots corresponding to ribosomal and nonribosomal proteins were excised with a cork borer and treated at 600 in capped glass vials with 0.5 ml of 30% H202. Radioactivity was determined in 10 ml of Aquasol (New England Nuclear Corp.). The relative synthetic rate of each protein, Ai, was calculated as described in the text. This is a sample of the data presented in Table 1.
We conclude that the synthesis of the ribosomal proteins is coordinately repressed at the restrictive temperature. Kinetics of Inhibition of Ribosomal Protein Synthesis. To provide some insight into the mechanism by which the coordinate control of ribosomal protein synthesis occurs, we have studied the kinetics of the inhibition of ribosomal protein synthesis when a mutant is shifted from the permissive to the restrictive temperature (Fig. 4). In the wild type, although the nonribosomal proteins continue to be made at a relatively normal rate, there is a transient inhibition of the synthesis of ribosomal proteins (Fig. 4a). This finding is consistent with earlier reports of a transient inhibition of ribosome synthesis after a temperature increase (3). In cells carrying the mutation in rna 2, the synthesis of nonribosomal proteins continues normally, whereas the syn-
C.
O
!.0 '1,
z
z
PO,
90
DI 40 60 TIME (MINUTES)
FIG. 3. Stability of ribosomal proteins synthesized at the permissive and restrictive temperatures. A culture of ts 368 was grown overnight in [14C]leucine; half of the culture was kept at 230, the other half was shifted to 330 for 60 min. Spheroplasts were then pulsed for 20 min with [3H]leucine and chased for 60 min with unlabeled leucine (1 mg/ml). Aliquots were removed during the pulse and the chase, Ribosomal protein synthesis was followed as described in Fig. 2. The data are presented as the 3H/14C ratio for ribosomal proteins no. 8 (0) and no. 18 (@).(--- -) 230; (- ) 330. Similar data were obtained for thirty of the proteins listed in Table 1.
1550
Biochemistry: Gorenstein and Warner
Proc. Nati. Acad. Sci. USA 73 (1976)
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