Current Genetics 1,912(1979)
current Genetics © by Springer-Verlag 1979
The Monocistronic Nature of Ribosomal Protein Genes in Yeast Charles Gorenstein* and Jonathan R. Warner
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA
Summary. The synthesis of thirty five ribosomal ,proteins has been studied in Saccharomyces cerevisiae using a temperature sensitive mutant blocked in the initiation of polypeptides. The synthesis of all the ribosomal proteins was nearly equally affected by the block. This absence of polarity, together with previous genetic and biochemical data, indicates that eukaryotic ribosomal proteins are coded by monocistronic mRNAs. Key words: rate of synthesis of protek.us - ts block of initiation - decay of polysomes
In prokaryotes the synthesis of a large number of ribosomal proteins is coordinately regulated at the level of transcription. The mechanisms which lead to such coordinate synthesis have been elegantly deduced in prokaryotes by a variety of techniques. Genetic mapping (Jaskunas et al., 1974), sensitivity to rifampicin (Dennis, 1974b), sensitivity to UV (Hirsch-Kauffman et al., 1975) as well as restriction enzyme analysis of cloned ribosomal protein genes (Nomura, 1976), have shown that most of the ribosomal protein genes are clustered in a few regions on the E. coli genome and are transcribed as a limited number of polycistronic mRNAs. In yeast, on the other hand, while the synthesis of 50 ribosomal proteins is coordinately regulated at the level of mRNA synthesis (Warner and Gorenstein, 1977), little or no evidence exists for clustering of genes or polycistronic mRNA. Genetic evidence based on 3 antibiotic resistant markers suggests that ribosomal protein genes Offprint requests to: J. R. Warner * Present address: The Johns Hopkins University, School of
Medicine, Department of Pharmacology, and Experimental Therapeutics, 725 North Wolfe Street, Baltimore, Maryland 21205, USA.
are scattered throughout the genome (Mortimer and Hawthorne, 1973; Skogerson et al., 1973; Schindler et al., 1974). In yeast, the size of the growing polypeptide chain is proportional to the size of its polysome, leading Petersen and McLaughlin (1973) to Conclude that there are few if any mRNAs coding for more than a single protein. However, it would have been difficult for them to detect polycistron!c mRNAs at a level of 10 to 15%. From anether perspective Shelman ~nd Stewart (1975) have provided strong genetic evidence that a second initiation on a single mRNA, such as required for a polycistronic mRNA, does not occur in yeast. Nevertheless, polycistronic mRNAs which synthesize a single long polypeptide, subsequently cleaved to form two or more completed proteins, would not be detected in either experiment. Mager and Planta (1976), by analyzing polypeptides completed in vitro from various sized polysomes, concluded that some yeast ribosomal proteins are coded by small, presumably monocistronic, mRNA molecules. However, the same group of workers (Mager et al., 1977), measuring the sensitivity of ribosomal protein synthesis to ultraviolet radiation, suggested that the genes for yeast ribosomal proteins are clustered and are transcribed as large polycistronic units. As a new approach to determining whether there exist polycistronic mRNA molecules coding for more than one ribosomal protein, we have studied the effect of a block on the initiation of protein synthesis. The rationale is that blocking initiation will have a polar effect on the translation of polycistronic mRNA: a protein coded near its 3' end will be translated by more ribosomes after the block than a protein at its 5' end. Strain ts 187 orS. cerevisiae was isolated by Hartwell and McLaughlin (t969), who concluded that, at the restrictive temperature of 36 °, it is defective in the initia-
O172-8083/79/0001/0009/$01.00
10
C. Gorenstein and J. R. Warner: Yeast Ribosomal Protein Synthesis
O25
0
BOTTOM
!,t
TOP BOTTOM
TOP
Fig. la-d. Kinetics of polysome decay in ts 187 at the restrictive temperature. Sheroplasts of ts 187 were maintained at 23 °C (a) and shifted to 36 °C for 1 min (b), 3 min (c) and 10 min (d). Aliquots were removed and 100 #g/ml cycloheximide added. The cells were lysed in 0.5 ml CFB (0.01 M NaC1, 0.01 M PIPES, 0.005 M MgCI2, 0.001 M Dithiothreitol, 0.01 spermidine HC1, pH 6.5) containing 0.1% saponin. The lysate was spun at 15,000 xg for 10 min and the supernate layered on top of a 7-47% w/w linear sucrose gradient containing CFB and centrifuged for 110 min at 36,000 rpm in a Beckman SW41 rotor at 4 °C. Centrifugation is from right to left. The gradients were collected through a Gilford recording spectrophotometer. Monosomes are indicated by the arrows
X 5'
00000 \
~V
Y
,f " ~ # ~ F h
J\
13x
5' M
ny
Fig. 2. A s s u m e t h a t n x and n y , the n u m b e r of r i b o s o m e s s y n t h e sizing proteins X and Y, are proportional to the size of X and of
Y respectively. If the cells are in a steady state, where ribosomal proteins do not turn over (Gorenstein and Warner, 1977), the amount of 14C uniformly labeled protein X is also proportional to the size of X and hence to n x. After a block in initiation, ribosomes engaged in translation will run off, completing translation of their nascent polypeptides. For a monocistronic mRNA or for a polycistronic mRNA of type 1, the incorporation of 3H into X nX
after a block in initiation is proportional to - - , since on average 2 each ribosome will make half a chain. Therefore theAi value for protein X (see Legend to Table 1): nx __
3H x A x~
-~ 2 =1/2, 14Cx -nX
and is thus independent of n x and of the size of X. However for a polycistronic mRNA of type 2 or 3, while A x ~ 1/2, ny --
2 A y o: - -
+
n X
, ny
since the Y messageis translated not only by its run-offribosomes, but also by all upstream ribosomes. For the simple case where nx=ny,Ay
o:
3/2 andAY - Ax
=
3
tion of polypeptide chains. Fig. 1 confirms this suggestion by showing the effect on the polysome profile of transferring ts 187 from 23 °C to 36 °C. Whithin one minute after the temperature shift (Fig. lb), the proportion of ribosomes in the monosome region begins to increase, and within three minutes (Fig. l c ) n e a r l y 100% of the ribosomes are found in the monosome region. Since the mean transit time for a ribosome in S. c e r e v i s i a e is approximately one minute (Petersen and McLaughlin, 1973), these data indicate that the block in initiation is effective within about thirty seconds. One can envision at least three types of polycistronic mRNAs: 1) polycistronic mRNA with multiple initiation and termination sites. In this case, ribosomes initiate at each cistron and upon completion of each polypeptide chain, the ribosomes are released. 2)Polycistronic mRNA with a single initiation site, but multiple termination sites. In this type, ribosomes initiate at the first cistron and after terminating the first polypeptide, cross over the intercistronic divide and reinitiate at the next available site. 3) Polycistronic mRNA with a single initiation and termination site. In this case, one large polypeptide is synthesized which is later cleaved to form two or more polypeptides. Consider a polycistronic mRNA consisting of two equal sized cistrons, coding for polypeptides X and Y, and translated by a maximum number of ribosomes nx and ny (Fig. 2). For a polycistronic mRNA of type 1, if the initiation of protein synthesis is blocked while ongoing polypeptides are completed, X and Y will be translated to the same extent: thus Ay/A x "~ 1. For type 2, Y will be translated by its ribosomes plus those of X: thus Ay/A x ~-- 3. For type 3, the carboxy terminal end of the precursor polypeptide will be labeled to a higher extent than the N-terminal end; thus, after processing, Ay/A x 3. Therefore measuring the synthesis of proteins after a block in initiation is very sensitive in detecting polycistronic mRNA of types 2 and 3, and would be more so for larger mRNAs. The synthesis of ribosomal and non-ribosomal proteins in ts 187 at 23 °C and 36 °C is shown in Table 1. The bottom line shows the incorporation into the total culture. In wild type ceils, incorporation at 36 ° is twice that at 23 ° (Gorenstein and Warner, 1976). In t s 187, incorporation at 36 ° is only one fourth that at 23 ° , indicating again the rapidity of the initiation block. At the restrictive temperature the relative rate of synthesis, Ai, of 35 ribosomal proteins decreases compared to that of nonribosomal proteins. However it is clear that the effect on all proteins is about the same. In particular the synthesis of no protein is substantially above the mean; there is little evidence of polarity. To substantiate this finding, cells were labeled thirty seconds after the temperature shift (Table 1C), which would exaggerate any polarity, as described in the analysis of Fig. 2. In this experiment,
C. Gorenstein and J. R. Warner: Yeast Ribosomal Protein Synthesis Table 1. Synthesis of ribosomal and nonribosomal proteins A culture of ts 187 was grown overnight at 23 °C in synthetic complete media (SC) containing 14C leucine (2.5 #Ci/ml). Three equal aliquots were removed and a) pulsed at 23 °C for 5 rain with 3H leucine (100 #Ci/ml), b) shifted to 36 °C and immediately pulsed for 5 min with 3H leucine, or c) shifted to 36 °C and after 30 s pulsed with 3H leucine for 5 rain. The incorporation of label was arrested by pouring the ceils into ice. Approximately 108 cells were suspended in 1 ml of distilled water. Enough glass beads were added (0.45 mm diameter) to cover the level of liquid and the cells were broken by vortexing for 1 rain. Immediately 0.1 vol 1 M MgCI2 and 2 volumes glacial acetic were added, the extract stirred for'30 min in ice, and precipitated RNA removed by centrifugation at 20,000 x g for 15 min. The supernate was dialyzed versus 1% acetic and lyophilized. 2D gel analysis of ribosomal proteins as well as the nomenclature used in identifying the proteins have been described previously (Gorenstein and Warner, 1976). The relative rate of synthesis of ribosomal and non-ribosomal proteins was measured according to the procedure of Dennis (1974a). After 2D gel electrophoresis of a whole cell extract, the 3H/14C ratio for each protein was compared to the 3H/14C ratio found in total protein. The relative rate of synthesis of the ith protein, Ai, is defined as
Ai=
3H/14C in the ith protein 3H/14C in total protein
the synthesis o f a small n u m b e r o f proteins, e.g. 16, 22, 40, were s o m e w h a t above the mean, but n o t sufficiently so as to suggest that any are synthesized on a polycistronic m R N A . While a polycistronic m R N A o f t y p e 1 is n o t formally e x c l u d e d by this e x p e r i m e n t , previous results (Petersen and McLaughlin, 1973; Sherman and Stewart, 1975) make it unlikely. F u r t h e r m o r e , for E. coli, Petersen et al. (1978) suggest that m o s t polycistronic m R N A is o f t y p e 2 rather than type 1, since ribosomes n o r m a l l y cross the intercistronic divide and initiate at the n e x t available site w i t h o u t dissociation. The e x p e r i m e n t presented here does n o t address the possibility that polycistronic nuclear R N A is synthesized and processed into monocistronic units prior to translation in the cytoplasm. Why the synthesis o f ribosomal proteins is specifically reduced in ts 187 is n o t k n o w n . H o w e v e r , this situation differs f r o m that seen in wild t y p e cells subjected to a t e m p e r a t u r e shift (Gorenstein and Warner, 1976 ; Warner and Gorenstein, 1977) in that the effect is i m m e d i a t e rather than requiring 20 min for full expression. Whether this indicates the inactivation o f specific initiation factors required for ribosomal protein synthesis m u s t await a m o r e detailed e x a m i n a t i o n o f the lesion in ts 187.
Acknowledgements. This work has been supported by grants from the NSF PCM 75-03938A01, the NIH1R01 GM 25532 and the ACS NP 72 I.
11
Ai
Ai
Ai
a) 23 °C
b) 36 °C
c) 36 °C (30 s delay)
Ribosomal protein 1 2 5 6 8
1.11 1.03 1.09 1.16 1.13
0.50 0.50 0.77 0.88 0.50
0.67 0.38 0.58 0.70 0.41
9 12 13 15 16
0.78 0.99 1.24 0.92 1.55
0.47 0.58 0.80 0.66 0.97
0.38 0.52 0.64 0.61 0.91
18 21 22 23 25
0.91 1.10 1.09 1.00 1.00
0.52 0.66 0.80 0.47 0.69
0.44 0.32 0.91 0.50 0.61
27 28 30 33 37
0.92 1.08 1.09 0.88 1.11
0.52 0.55 0.55 0.58 0.97
0.44 0.50 0.47 0.44 0.85
38 39 40 41 44
1.13 1.00 1.18 0.81 0.99
0.80 0.58 0.80 0.72 0.66
0.76 0.50 0.91 0.70 0.64
45 46 47 49 50
0.92 0.94 0.98 0.90 0.98
0.80 0.86 0.77 0.77 0.83
0.73 0.76 0.76 0.79 0.82
52 61 62 63 64
0.95 0.95 0.89 0.91 0.91
0.77 0.69 0.72 0.58 0.69
0.76 0.52 0.67 0.70 0.58
Mean
1.018
0.68
0.62
Nonribosomal protein A B C D E F G
1.05 1.79 1.12 1.56 1.71 1.21 1.44
1.11 1.69 1.27 1.15 1.52 1.33 1.47
0.91 1.29 1.14 0.82 1.41 1.44 1.47
Mean
1.41
1.36
1.21
Total 3H/14C
1.48
0.37
0.34
12 References Dennis, P. P.: J. Mol. Biol. 89, 223-232 (1974) Dennis, P. P.: Mol. Gen. Genet. 134, 39-47 (1974b) Gorenstein, C., Warner, J. R.: Proc. Nat. Acad. Sci. U.S.A. 73, 1547-1551 (1976) Hartwell, L., McLaughlin, C. S.: Proc. Nat. Acad. Sci. U.S.A. 62, 468 (1969) Hartwell, L., McLaughlin, C., Warner, J. R.: Mol. Gen. Genet. 109, 42-56 (1970) Hirsch-Kauffman, M., Schweiger, M., Herrlich, P., Ponta, H., Rahmsdorf, H., Pai, S., Wittman, H.: Eur. J. Biochem. 52, 469-475 (1975) Jaskunas, S. R., Nomura, M., Davies, J.: In: Ribosomes. Nomura, M., Tissieres, A., Lengyel, P., (eds.), pp. 333-368. New York: Cold Spring Harbor Laboratory (1974) Mager, W. H., Planta, R. J.: Eur. J. Biochem. 62, 193-197 (1976) Mager, W. H., Retal, J., Planta, R. J., Bollen, H. P. M. G., DeRegt, C. H. F. V., Hoving, H.: Eur. J. Biochem. 78, 575-583 (1977) Mortimer, R. K., Hawthorne, D. C.: Genetics 74, 33-54 (1973)
C. Gorenstein and J. R. Warner: Yeast Ribosomal Protein Synthesis Nomura, M.: Cell 9, 633-644 (1976) Petersen, N. S., McLaughlin, C. S.: J. Mol. BioL 81, 33-45 (1973) Petersen, H. U., Joseph, E., Ullman, A., Danchin, A.: J. BacterioL 135,453-459 (1978) Schindler, D., Grant, P., Davies, J.: Nature (London) 248, 535536 (1974) Sherman, F., Stewart, J. W.: The Use of Iso-l-Cytochrome c Mutants of Yeast for Elucidating the Nucleotide Sequences that Govern Initiation of Translation. Proceedings of the 10th FEBS Meeting, pp. 175-191 (1975) Skogerson, L.,. McLaughlin, C., Wakatama, E.: J. Bacteriol. 116, 818-822 (1973) Warner, J. R., Gorenstein, C.: Cell 11,201-212 (1977)
C o m m u n i c a t e d b y F. Kaudewitz
Received April 23, 1979
current Genetics © by Springer-Verlag 1979
The Monocistronic Nature of Ribosomal Protein Genes in Yeast Charles Gorenstein* and Jonathan R. Warner
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA
Summary. The synthesis of thirty five ribosomal ,proteins has been studied in Saccharomyces cerevisiae using a temperature sensitive mutant blocked in the initiation of polypeptides. The synthesis of all the ribosomal proteins was nearly equally affected by the block. This absence of polarity, together with previous genetic and biochemical data, indicates that eukaryotic ribosomal proteins are coded by monocistronic mRNAs. Key words: rate of synthesis of protek.us - ts block of initiation - decay of polysomes
In prokaryotes the synthesis of a large number of ribosomal proteins is coordinately regulated at the level of transcription. The mechanisms which lead to such coordinate synthesis have been elegantly deduced in prokaryotes by a variety of techniques. Genetic mapping (Jaskunas et al., 1974), sensitivity to rifampicin (Dennis, 1974b), sensitivity to UV (Hirsch-Kauffman et al., 1975) as well as restriction enzyme analysis of cloned ribosomal protein genes (Nomura, 1976), have shown that most of the ribosomal protein genes are clustered in a few regions on the E. coli genome and are transcribed as a limited number of polycistronic mRNAs. In yeast, on the other hand, while the synthesis of 50 ribosomal proteins is coordinately regulated at the level of mRNA synthesis (Warner and Gorenstein, 1977), little or no evidence exists for clustering of genes or polycistronic mRNA. Genetic evidence based on 3 antibiotic resistant markers suggests that ribosomal protein genes Offprint requests to: J. R. Warner * Present address: The Johns Hopkins University, School of
Medicine, Department of Pharmacology, and Experimental Therapeutics, 725 North Wolfe Street, Baltimore, Maryland 21205, USA.
are scattered throughout the genome (Mortimer and Hawthorne, 1973; Skogerson et al., 1973; Schindler et al., 1974). In yeast, the size of the growing polypeptide chain is proportional to the size of its polysome, leading Petersen and McLaughlin (1973) to Conclude that there are few if any mRNAs coding for more than a single protein. However, it would have been difficult for them to detect polycistron!c mRNAs at a level of 10 to 15%. From anether perspective Shelman ~nd Stewart (1975) have provided strong genetic evidence that a second initiation on a single mRNA, such as required for a polycistronic mRNA, does not occur in yeast. Nevertheless, polycistronic mRNAs which synthesize a single long polypeptide, subsequently cleaved to form two or more completed proteins, would not be detected in either experiment. Mager and Planta (1976), by analyzing polypeptides completed in vitro from various sized polysomes, concluded that some yeast ribosomal proteins are coded by small, presumably monocistronic, mRNA molecules. However, the same group of workers (Mager et al., 1977), measuring the sensitivity of ribosomal protein synthesis to ultraviolet radiation, suggested that the genes for yeast ribosomal proteins are clustered and are transcribed as large polycistronic units. As a new approach to determining whether there exist polycistronic mRNA molecules coding for more than one ribosomal protein, we have studied the effect of a block on the initiation of protein synthesis. The rationale is that blocking initiation will have a polar effect on the translation of polycistronic mRNA: a protein coded near its 3' end will be translated by more ribosomes after the block than a protein at its 5' end. Strain ts 187 orS. cerevisiae was isolated by Hartwell and McLaughlin (t969), who concluded that, at the restrictive temperature of 36 °, it is defective in the initia-
O172-8083/79/0001/0009/$01.00
10
C. Gorenstein and J. R. Warner: Yeast Ribosomal Protein Synthesis
O25
0
BOTTOM
!,t
TOP BOTTOM
TOP
Fig. la-d. Kinetics of polysome decay in ts 187 at the restrictive temperature. Sheroplasts of ts 187 were maintained at 23 °C (a) and shifted to 36 °C for 1 min (b), 3 min (c) and 10 min (d). Aliquots were removed and 100 #g/ml cycloheximide added. The cells were lysed in 0.5 ml CFB (0.01 M NaC1, 0.01 M PIPES, 0.005 M MgCI2, 0.001 M Dithiothreitol, 0.01 spermidine HC1, pH 6.5) containing 0.1% saponin. The lysate was spun at 15,000 xg for 10 min and the supernate layered on top of a 7-47% w/w linear sucrose gradient containing CFB and centrifuged for 110 min at 36,000 rpm in a Beckman SW41 rotor at 4 °C. Centrifugation is from right to left. The gradients were collected through a Gilford recording spectrophotometer. Monosomes are indicated by the arrows
X 5'
00000 \
~V
Y
,f " ~ # ~ F h
J\
13x
5' M
ny
Fig. 2. A s s u m e t h a t n x and n y , the n u m b e r of r i b o s o m e s s y n t h e sizing proteins X and Y, are proportional to the size of X and of
Y respectively. If the cells are in a steady state, where ribosomal proteins do not turn over (Gorenstein and Warner, 1977), the amount of 14C uniformly labeled protein X is also proportional to the size of X and hence to n x. After a block in initiation, ribosomes engaged in translation will run off, completing translation of their nascent polypeptides. For a monocistronic mRNA or for a polycistronic mRNA of type 1, the incorporation of 3H into X nX
after a block in initiation is proportional to - - , since on average 2 each ribosome will make half a chain. Therefore theAi value for protein X (see Legend to Table 1): nx __
3H x A x~
-~ 2 =1/2, 14Cx -nX
and is thus independent of n x and of the size of X. However for a polycistronic mRNA of type 2 or 3, while A x ~ 1/2, ny --
2 A y o: - -
+
n X
, ny
since the Y messageis translated not only by its run-offribosomes, but also by all upstream ribosomes. For the simple case where nx=ny,Ay
o:
3/2 andAY - Ax
=
3
tion of polypeptide chains. Fig. 1 confirms this suggestion by showing the effect on the polysome profile of transferring ts 187 from 23 °C to 36 °C. Whithin one minute after the temperature shift (Fig. lb), the proportion of ribosomes in the monosome region begins to increase, and within three minutes (Fig. l c ) n e a r l y 100% of the ribosomes are found in the monosome region. Since the mean transit time for a ribosome in S. c e r e v i s i a e is approximately one minute (Petersen and McLaughlin, 1973), these data indicate that the block in initiation is effective within about thirty seconds. One can envision at least three types of polycistronic mRNAs: 1) polycistronic mRNA with multiple initiation and termination sites. In this case, ribosomes initiate at each cistron and upon completion of each polypeptide chain, the ribosomes are released. 2)Polycistronic mRNA with a single initiation site, but multiple termination sites. In this type, ribosomes initiate at the first cistron and after terminating the first polypeptide, cross over the intercistronic divide and reinitiate at the next available site. 3) Polycistronic mRNA with a single initiation and termination site. In this case, one large polypeptide is synthesized which is later cleaved to form two or more polypeptides. Consider a polycistronic mRNA consisting of two equal sized cistrons, coding for polypeptides X and Y, and translated by a maximum number of ribosomes nx and ny (Fig. 2). For a polycistronic mRNA of type 1, if the initiation of protein synthesis is blocked while ongoing polypeptides are completed, X and Y will be translated to the same extent: thus Ay/A x "~ 1. For type 2, Y will be translated by its ribosomes plus those of X: thus Ay/A x ~-- 3. For type 3, the carboxy terminal end of the precursor polypeptide will be labeled to a higher extent than the N-terminal end; thus, after processing, Ay/A x 3. Therefore measuring the synthesis of proteins after a block in initiation is very sensitive in detecting polycistronic mRNA of types 2 and 3, and would be more so for larger mRNAs. The synthesis of ribosomal and non-ribosomal proteins in ts 187 at 23 °C and 36 °C is shown in Table 1. The bottom line shows the incorporation into the total culture. In wild type ceils, incorporation at 36 ° is twice that at 23 ° (Gorenstein and Warner, 1976). In t s 187, incorporation at 36 ° is only one fourth that at 23 ° , indicating again the rapidity of the initiation block. At the restrictive temperature the relative rate of synthesis, Ai, of 35 ribosomal proteins decreases compared to that of nonribosomal proteins. However it is clear that the effect on all proteins is about the same. In particular the synthesis of no protein is substantially above the mean; there is little evidence of polarity. To substantiate this finding, cells were labeled thirty seconds after the temperature shift (Table 1C), which would exaggerate any polarity, as described in the analysis of Fig. 2. In this experiment,
C. Gorenstein and J. R. Warner: Yeast Ribosomal Protein Synthesis Table 1. Synthesis of ribosomal and nonribosomal proteins A culture of ts 187 was grown overnight at 23 °C in synthetic complete media (SC) containing 14C leucine (2.5 #Ci/ml). Three equal aliquots were removed and a) pulsed at 23 °C for 5 rain with 3H leucine (100 #Ci/ml), b) shifted to 36 °C and immediately pulsed for 5 min with 3H leucine, or c) shifted to 36 °C and after 30 s pulsed with 3H leucine for 5 rain. The incorporation of label was arrested by pouring the ceils into ice. Approximately 108 cells were suspended in 1 ml of distilled water. Enough glass beads were added (0.45 mm diameter) to cover the level of liquid and the cells were broken by vortexing for 1 rain. Immediately 0.1 vol 1 M MgCI2 and 2 volumes glacial acetic were added, the extract stirred for'30 min in ice, and precipitated RNA removed by centrifugation at 20,000 x g for 15 min. The supernate was dialyzed versus 1% acetic and lyophilized. 2D gel analysis of ribosomal proteins as well as the nomenclature used in identifying the proteins have been described previously (Gorenstein and Warner, 1976). The relative rate of synthesis of ribosomal and non-ribosomal proteins was measured according to the procedure of Dennis (1974a). After 2D gel electrophoresis of a whole cell extract, the 3H/14C ratio for each protein was compared to the 3H/14C ratio found in total protein. The relative rate of synthesis of the ith protein, Ai, is defined as
Ai=
3H/14C in the ith protein 3H/14C in total protein
the synthesis o f a small n u m b e r o f proteins, e.g. 16, 22, 40, were s o m e w h a t above the mean, but n o t sufficiently so as to suggest that any are synthesized on a polycistronic m R N A . While a polycistronic m R N A o f t y p e 1 is n o t formally e x c l u d e d by this e x p e r i m e n t , previous results (Petersen and McLaughlin, 1973; Sherman and Stewart, 1975) make it unlikely. F u r t h e r m o r e , for E. coli, Petersen et al. (1978) suggest that m o s t polycistronic m R N A is o f t y p e 2 rather than type 1, since ribosomes n o r m a l l y cross the intercistronic divide and initiate at the n e x t available site w i t h o u t dissociation. The e x p e r i m e n t presented here does n o t address the possibility that polycistronic nuclear R N A is synthesized and processed into monocistronic units prior to translation in the cytoplasm. Why the synthesis o f ribosomal proteins is specifically reduced in ts 187 is n o t k n o w n . H o w e v e r , this situation differs f r o m that seen in wild t y p e cells subjected to a t e m p e r a t u r e shift (Gorenstein and Warner, 1976 ; Warner and Gorenstein, 1977) in that the effect is i m m e d i a t e rather than requiring 20 min for full expression. Whether this indicates the inactivation o f specific initiation factors required for ribosomal protein synthesis m u s t await a m o r e detailed e x a m i n a t i o n o f the lesion in ts 187.
Acknowledgements. This work has been supported by grants from the NSF PCM 75-03938A01, the NIH1R01 GM 25532 and the ACS NP 72 I.
11
Ai
Ai
Ai
a) 23 °C
b) 36 °C
c) 36 °C (30 s delay)
Ribosomal protein 1 2 5 6 8
1.11 1.03 1.09 1.16 1.13
0.50 0.50 0.77 0.88 0.50
0.67 0.38 0.58 0.70 0.41
9 12 13 15 16
0.78 0.99 1.24 0.92 1.55
0.47 0.58 0.80 0.66 0.97
0.38 0.52 0.64 0.61 0.91
18 21 22 23 25
0.91 1.10 1.09 1.00 1.00
0.52 0.66 0.80 0.47 0.69
0.44 0.32 0.91 0.50 0.61
27 28 30 33 37
0.92 1.08 1.09 0.88 1.11
0.52 0.55 0.55 0.58 0.97
0.44 0.50 0.47 0.44 0.85
38 39 40 41 44
1.13 1.00 1.18 0.81 0.99
0.80 0.58 0.80 0.72 0.66
0.76 0.50 0.91 0.70 0.64
45 46 47 49 50
0.92 0.94 0.98 0.90 0.98
0.80 0.86 0.77 0.77 0.83
0.73 0.76 0.76 0.79 0.82
52 61 62 63 64
0.95 0.95 0.89 0.91 0.91
0.77 0.69 0.72 0.58 0.69
0.76 0.52 0.67 0.70 0.58
Mean
1.018
0.68
0.62
Nonribosomal protein A B C D E F G
1.05 1.79 1.12 1.56 1.71 1.21 1.44
1.11 1.69 1.27 1.15 1.52 1.33 1.47
0.91 1.29 1.14 0.82 1.41 1.44 1.47
Mean
1.41
1.36
1.21
Total 3H/14C
1.48
0.37
0.34
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C. Gorenstein and J. R. Warner: Yeast Ribosomal Protein Synthesis Nomura, M.: Cell 9, 633-644 (1976) Petersen, N. S., McLaughlin, C. S.: J. Mol. BioL 81, 33-45 (1973) Petersen, H. U., Joseph, E., Ullman, A., Danchin, A.: J. BacterioL 135,453-459 (1978) Schindler, D., Grant, P., Davies, J.: Nature (London) 248, 535536 (1974) Sherman, F., Stewart, J. W.: The Use of Iso-l-Cytochrome c Mutants of Yeast for Elucidating the Nucleotide Sequences that Govern Initiation of Translation. Proceedings of the 10th FEBS Meeting, pp. 175-191 (1975) Skogerson, L.,. McLaughlin, C., Wakatama, E.: J. Bacteriol. 116, 818-822 (1973) Warner, J. R., Gorenstein, C.: Cell 11,201-212 (1977)
C o m m u n i c a t e d b y F. Kaudewitz
Received April 23, 1979
