JOURNAL OF BACTERIOLOGY, June 1975, p. 855-865 Copyright 0 1975 American Society for Microbiology

Vol. 122, No. 3 Printed in U.S.A.

Effect of Growth Rate on the Amounts of Ribosomal and Transfer Ribonucleic Acids in Yeast CLIVE WALDRON AND FRANQOIS LACROUTE Laboratoire de Genetique Physiologique, I.B.M.C., 15 rue Descartes, 67000 Strasbourg, France Received for publication 25 February 1975

The steady-state growth rate of Saccharomyces cerevisiae was varied by growing the cells in different media. The total amount of ribonucleic acid (RNA) per cell was found to decrease as a nonlinear function of decreasing growth rate. The RNA from cells growing in different media was analyzed by polyacrylamide gel electrophoresis. Although the amounts of both ribosomal RNA and transfer RNA decreased with decreasing growth rate, the ratio of ribosomal to transfer RNA was not constant. As the growth rate was reduced the ribosomal RNA fraction decreased slightly, whereas the transfer RNA fraction increased slightly. Thus the levels of ribosomal and transfer RNA were regulated to similar yet different extents. The levels of the different ribosomal RNA species were more closely coordinated. At all growth rates the ribosomal RNAs (including 5S RNA) were present in equimolar amounts. The rate of protein synthesis in yeast cells also decreased with decreasing growth rate. The low rates of protein synthesis did not appear to be due to limiting numbers of ribosomes or transfer RNA molecules. In bacteria, the cellular content of ribonucleic acid (RNA) decreases with decreasing growth rates (3, 13, 16). The RNA consists of mainly two types: the bulk is ribosomal RNA (rRNA) and most of the remainder is transfer RNA (tRNA). With decreasing growth rates the rRNA fraction of total RNA decreases slightly, but the tRNA fraction increases (4, 8, 27). The net result of these changes is that with decreasing growth rates the cellular contents of both rRNA and tRNA decrease, but to slightly different extents. The protein content of bacterial cells also decreases with growth rate, but in such a way that the rate of protein synthesis per unit of rRNA is constant over a wide range of growth rates (3, 9, 16). Thus ribosome efficiency is largely independent of growth rate in bacteria. Total RNA content also decreases with decreasing growth rates in a number of eukaryotic microorganisms, namely, the yeast Saccharomyces cerevisiae (17, 26, 32), the protozoan Tetrahymena pyriformis (14), the alga Prototheca zopfii (22), and the slime mold Physarum polycephalum (B. S. Plaut and G. Turnock, personal communication). The effect of growth rate on the relative amounts of rRNA and tRNA have been studied only in T. pyriformis and P. polycephalum. The results obtained are similar to those from bacteria: the two types of RNA decrease to slightly different

extents with decreasing growth rate (14; Plaut and Tumock, personal communication). In these organisms the net rate of protein synthesis per unit of rRNA decreases as the cells grow more slowly. Thus it appears that in eukaryotic microorganisms, in contrast to bacteria, ribosome efficiency is dependent on growth rate. S. cerevisiae is particularly suitable for the study of RNA and protein synthesis in eukaryotes. It is a non-differentiating, unicellular yeast whose growth rate can be varied over a wide range by changing the composition of the growth medium. Furthermore, many mutants of S. cerevisiae that are altered in RNA or protein synthesis have been isolated (11). Nevertheless, there have been few studies of growth rate-dependent changes in the macromolecular composition of yeasts. The most extensive analyses are limited to slow-growing chemostat cultures (17) or to cells growing in three media (32). In neither case was it demonstrated that the cells were in balanced growth, nor were the relative amounts of rRNA and tRNA examined. This study involves six different media producing a large range of steadystate growth rates. Using these media, we investigated the effects of growth rate on the levels of rRNA and tRNA in yeast cells. We also studied the efficiency with which the rRNA is used in protein synthesis in yeast cells growing in different media.

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WALDRON AND LACROUTE

MATERIALS AND METHODS Organism and growth conditions. All experiments were performed with S. cerevisiae strain FL 521 (ura2; a). This strain carries a deletion in the structural gene for aspartate transcarbamylase (M. L. Bach, personal communication) and therefore cannot synthesize any pyrimidine endogenously. The strain will grow when provided with uracil or cytosine, since yeast synthesizes all the pyrimidine nucleotides from either base (R. Jund, Ph.D. thesis, Universite Louis Pasteur, Strasbourg, 1973). The six media used are listed in Table 1. Cultures (10 ml) of liquid medium were incubated at 30 C in Klett flasks on a reciprocal shaker. The nutritionally poor media L and E were inoculated with samples of a liquid medium Y culture containing cells in late log phase. The other media were inoculated from colonies growing on solid medium. Growth of liquid cultures was monitored turbidimetrically in a Klett-Summerson colorimeter, using a red filter (about 670 nm) for medium C and a blue filter (about 430 nm) for the other media. In the range of 20 to 100 Klett units (either filter), cultures grew at exponential rates characteristic of each medium (Table 1). Their turbidity was not allowed to exceed 100 Klett units, even though exponential growth was maintained in all media except L until much higher turbidities were reached. In medium L, exponential growth was not maintained beyond 80 Klett units (blue filter). Each growth rate (j) was calculated as the reciprocal of the generation time in hours. Determination of dry weights, cell number, and protein content. For dry weight estimation, membrane filters (0.80-Mm pore size; Millipore Corp.) were first soaked in distilled water and then dried and weighed. The cells in 8 ml of culture were collected on these prewashed filters, rinsed with water, and then dried at 100 C to constant weight. It was found that a culture turbidity of 1 Klett unit (red filter) in medium C is equivalent to 5.13 tsg (dry weight) per ml. Similarly, each Klett unit (blue filter) in all the other media is equivalent to 3.55 ug (dry weight) per ml. Cell counts were made by using a hemocytometer slide. The relationship between turbidity and cell number was not constant but depended on the growth

J. BACTERIOL.

rate of the cells and on which filter was used for turbidity estimations. The equivalence of turbidity and cell number can be calculated for any given culture from the growth rate of the culture, the relationship between turbidity and dry weight (see above), and the dry weight per cell at different growth rates (see Fig. 3A). For example, in a culture growing in medium Y with a generation time of 2 h, the average dry weight per cell is 14.4 pg, and therefore each Klett unit (blue filter) is equivalent to about 2.5 105 cells per ml. The protein content of acidprecipitated cells was determined by the method of Lowry et al. (15), using bovine serum albumin (fraction V; Sigma Chemical Co.) as standard. These values were not corrected for any difference between yeast protein and serum albumin in reactivity with the Folin reagent. Although this difference may be quite large, it should not affect our comparison of cellular protein content in different media. Estimations of nucleic acid content. Total nucleic acid was determined from the amount of radioactive uracil incorporated into acid-insoluble material. Cultures were grown for several generations in the presence of [14Cluracil (0.5 ACi/ml; 10.11 ug/ml). At intervals throughout the exponential growth phase, 0.2-ml samples of culture were mixed with 2 ml of ice-cold 5% trichloroacetic acid. After 1 h in the cold, the precipitated material was collected on membrane filters (0.80-Mm pore size) and washed three times with 10 ml of cold 5% trichloroacetic acid and once with 5 ml of cold water. The filters were dried and then mixed with 10 ml of toluene containing 0.5% (wt/vol) 2,5-diphenyloxazole (PPO). Radioactivity was measured in an Intertechnique SL 30 spectrometer. In these experiments almost all of the acid-insoluble radioactivity was present as cytidine and uridine residues of RNA, both pyrimidines being derived from the exogenously supplied uracil (R. Jund, Ph.D. thesis). Given that these pyrimidines constitute 43.8% (by weight) of bulk yeast RNA (19), the quantity of RNA in cells can be calculated from the amount of uracil incorporated (Mg of RNA 6.24 x Ag of uracil incorporated). Nucleic acid content was also determined by the method of Von Meyenburg (30), in which the products x

=

TABLE 1. Composition of growth mediaa Nitrogen sourceRange growth rates ce Nit (generations/h)

of

Medium

Carbon source"

C Y F P L E

Glucose Glucose Fructose Glucose Lactose Ethanol

Casein hydrolysate (10) Ammonium sulfate (5) Ammonium sulfate (5) L-Proline (2) Ammonium sulfate (5) Ammonium sulfate (5)

0.53-0.72 0.38-0.54 0.38-0.45 0.20-0.29 0.057-0.088 0.035-0.079

Avg growth Avg generarate

(iU)

tion time (h)

0.62 0.46 0.41 0.25

1.6 2.1 2.4 4.0 13.2 17.9

0.076 0.056

a All media contained uracil at a concentration of 10 gg/ml. For all media except P, the carbon source (20 g/liter) was added to a solution containing (per liter) 6.7 g of yeast nitrogen base without amino acids (Difco). Casein hydrolysate was also added to this solution when making medium C. The supplements for medium P were added to a solution containing (per liter) 1.45 g of yeast nitrogen base without amino acids or ammonium (Difco).

RNA COMPOSITPION IN YEAST

VOL. 122, 1975

of selective hydrolysis are estimated spectrophotometrically. The cells in 8 ml of culture were precipitated in the cold for 30 min with 1.5 ml of 1.2 N perchloric acid (PCA), collected by centrifugation, and washed with 5 ml of 0.2 N PCA. After again collecting the cells by centrifugation, the pellet was treated with 2 ml of 0.3 N NaOH for 1 h at 37 C. This treatment is sufficient to hydrolyze all the RNA, since in control experiments 97% of radioactivity due to incorporated [14C ]uracil was rendered acid soluble by the procedure (data not shown). After hydrolysis, the cell suspension was mixed with 1 ml of 1.2 N PCA, left for 20 min in the cold, and centrifuged at 26,000 x g for 20 min. The supernatant contained the products of RNA hydrolysis, the amounts of which could be determined from the absorbance at 260 nm (1 unit of absorbance 31.1 Ag of yeast RNA/ml). This relationship was calculated from the extinction coefficients in acid solution (1) of a mixture of ribonucleoside monophosphates corresponding to the composition of bulk yeast RNA (19). A deoxyribonucleic acid (DNA) assay was performed on the pellet of acidinsoluble material after washing with 2 ml of 0.2 N PCA and recentrifugation. This final pellet was dissolved in 0.1 ml of 0.06 N NaOH and treated for 3 h at 25 C with 50 Mg of deoxyribonuclease I in 0.9 ml of TSM buffer [10 mM tris(hydroxymethyl)aminomethane, 4 mM succinic acid, 10 mM magnesium acetate, pH 7.4]. The suspension was then mixed with 0.5 ml of 0.2 N PCA, left for 30 min in the cold, and centrifuged (26,000 x g for 20 min). The quantities of the products of DNA hydrolysis in the supernatant were estimated from the absorbance at 260 nm (1 unit of absorbance 29.4 Mg of yeast DNA/ml). This relationship was calculated from the extinction coefficients in acid solution (1) of a mixture of deoxyribonucleoside monophosphates corresponding to the composition of yeast DNA (18). RNA extraction. Cultures were labeled for at least three generations in medium containing [14C Juracil (0.5 MCi/ml; 11 Mg/ml). When the cultures reached a density corresponding to about 300 gg (dry weight)/ ml, 3.5-ml samples were mixed with 7 ml of ice-cold 96% ethanol to stop further RNA synthesis. In the case of medium L this density corresponds to late log phase, and therefore larger volumes of L cultures were taken at lower cell density and mixed with twice their volumes of ethanol. Thus the same total dry weight of exponentially growing cells was extracted in all experiments. The ethanol-arrested cells were collected by centrifugation and washed by resuspension in 5 ml of acetate buffer (1 mM sodium acetate, 5 mM NaCl, and 0.1 mM magnesium acetate, pH 5.1) containing 66% ethanol. After recentrifugation, the pellets were resuspended in 1.2 ml of acetate buffer containing 0.5% (wt/vol) sodium dodecyl sulfate (SDS). The cells were broken by treatment with glass beads (0.5-mm diameter) in a Chemap Vibromix (Chemap A.E., Mannedorf, Switzerland). A further 1.2 ml of acetate buffer was added to the broken cells, followed by 2.4 ml of organic mixture (500 ml of buffer-saturated phenol, 70 ml of m-cresol, and 0.5 g of 8-hydroxyquinoline). Deproteinization was then achieved by vortexing the mixture for 5 min at room =

=

857

temperature. The phases were separated by centrifugation, and to the aqueous phase was added 200 ,g of Torula RNA (Sigma) as carrier and 2 volumes of 96% ethanol containing 0.2 M sodium acetate. After overnight precipitation at -20 C, the RNA was collected by centrifugation, resuspended in 2.5 ml of acetate buffer containing 0.5% (wt/vol) SDS, and reprecipitated with ethanol-sodium acetate. After the second precipitation, the RNA was collected by centrifugation, dried under vacuum, and stored at - 30 C. Except for the deproteinization step, all stages of this extraction were performed at 0 to 4 C to minimize ribonuclease activity. For the same reason all glassware was acid washed and sterilized and all buffers were sterile. By determining the yield of acid-insoluble radioactivity from cells grown in the presence of [14C]uracil, we estimate that the efficiency of RNA extraction is about 85%. In certain experiments RNA was extracted from cells labeled with both [3H]uracil and ["4Cicytosine. When comparing the 3H/14C ratio in the purified RNA with that in the cells, we used the protocol of Dennis and Bremer (2) to exclude the problem of differential quenching of 'H radioactivity (within cells). Samples of the original ethanol-arrested cells, and of the RNA preparations made from them, were precipitated with cold 5% trichloroacetic acid and acid-insoluble material collected on Whatman GF/C filters. The filters were incubated overnight at 30 C in 2 ml of 0.3 N NaOH to hydolyze RNA. The resulting solutions were cooled to 0 C and mixed with 1 ml of 1.2 N PCA in the cold. Insoluble material was removed by filtration through membrane filters (0.45-Mm pore size), and 1 ml of filtrate was mixed with 10 ml of scintillation fluid (33% [vol/vol] Triton X-100, 67% [vol/vol I toluene, 0.5% [vol/vol] PPO) and then counted. By this procedure the radioactivities of both cells and RNA preparations are counted under identical conditions as soluble ribonucleotides. Polyacrylamide gel electrophoresis. Slabs of gel (18 by 14 by 0.3 cm), containing 2.5% polyacrylamide strengthened with 0.5% agarose (20) or only 7.5% polyacrylamide, were made as described by De Wachter and Fiers (5). The gels were prepared in the absence of SDS from acrylamide and bisacrylamide (19:1, wt/wt) in TEB buffer (90 mM tris(hydroxymethyl)aminomethane-borate, 2.5 mM ethylenediaminetetraacetate, pH 8.3). N,N,N',N'-tetramethylethylenediamine (0.033 ml/g of acrylamide) and freshly dissolved ammonium persulfate (3.3%, wt/vol; 0.1 mVg of acrylamide) were used as catalysts. Polymerization was allowed to proceed overnight at 20 C, after which the gels were prerun at 250 V for at least 1 h at 4 C, using an electrophoresis buffer of TEB with 0.1% (wt/vol) SDS. This prerun serves to remove unpolymerized acrylamides and catalysts from the gel and enables SDS to be introduced into the gel. Electrophoresis without SDS resulted in highly irreproducible yields of the smaller RNA species. After the prerun, the buffer was replaced with fresh TEB containing 0.1% SDS, and up to five 10-Mgl samples were applied to the gel. Each sample contained 5 to 10 ,g of RNA dissolved in electrophoresis buffer with 10% (wt/vol) sucrose and 0.05% (wt/vol)

858

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WALDRON AND L.CROUTE

bromophenol blue. Electrophoresis was at 250 V (about 20 mA/gel) at 4 C until the bromophenol blue marker had migrated about 8 cm from the origin (3 to 5 h, depending on the gel concentration). Immediately after electrophoresis, gels were dried down onto filter paper in a press at 80 C in a vacuum oven. To determine the distribution of radioactivity in a gel, pieces of the dried gel (2 by 10 mm) were incubated overnight at 37 C with 0.5 ml of 0.3 M NaOH to hydrolyze the RNA. The solution was subsequently neutralized with 0.5 ml of 0.2 N o-phosphoric acid and mixed with 10 ml of Triton-toluenePPO scintillation fluid. With this procedure over 90% of the applied counts can be detected in the gel. For calculating amounts of RNA from radioactivity in gels, the following pyrimidine contents (by weight) were used: 43.9% rRNA (19), 45.6% 5S RNA (12), and 48.5% 4S RNA (19). In calculating the number of RNA molecules, the following molecular weights were assumed: 1.3 x 106 for 25S RNA (29), 7 x 105 for 18S RNA (29), 5 x 104 for 5.8S RNA (24), 4 x 104 for 5S RNA (12), and 2.5 x 104 for 4S RNA. Chemicals. [2-4C Juracil (specific activity about 50 mCi/mmol), [2-14C ]cytosine (specific activity about 55 mCi/mmol), and [5-'Hjuracil (specific activity about 24 Ci/mmol) were obtained from CEA-France. Agarose for electrophoresis was from Merck, and acrylamide (purum) was from Fluka A. G. N,N'methylene bisacrylamide (Fluka practicum) was recrystallized from acetone before use. All other reagents were of analytical grade.

RESULTS Demonstration of steady states of growth in different media. For this study we wished to analyze only cells that were fully adapted to the medium in which they were growing. To demonstrate that our cultures were in such equilibrium, we investigated two criteria by which steady-state growth can be defined (16). The first is that growth will be exponential at a constant rate. Hence, the amount of any cell component should increase exponentially with time. The second criterion is that growth will be balanced, and therefore the amounts of different cell components should be maintained in constant ratios to one another. Macromolecular composition as a function of culture turbidity was studied in the range of 25 to 100 Klett units (either filter) for all media except L. Cultures in these media were harvested for RNA analysis at 80 (blue filter) or 60 (red filter) Klett units. For medium L, where cultures were harvested at only 60 Klett units (blue filter), cell composition was determined in the range of 20 to 80 Klett units (blue filter). Within these ranges cell number, turbidity, and RNA content (as measured by isotope incorporation) all increased exponentially at a rate characteristic of each medium (Fig. 1). To demonstrate constant ratios between other cell

8

E

4

C

E

8

2

' 12

TO

120

E 0

so

6

30

., 15 2

4

Time

6

(h)

FIG. 1. Example of exponential growth of a yeast culture. For this experiment cells were grown in medium F in the presence of radioactive uracil. Symbols: (0) RNA content (expressed as radioactivity incorporated into acid-insoluble material); (A) cell number; (0) culture turbidity.

components, we measured the dry weight, protein, or nucleic acid contents of cultures. For each medium four or five parallel cultures were harvested at different turbidities. All the parameters were directly proportional to turbidity within the ranges studied (Fig. 2). Thus the ratios of these cell components to one another were constant during exponential growth in any one medium. We conclude that in our experiments cultures were in balanced, steady-state growth when harvested for RNA analysis. Effects of growth rate on macromolecular composition. Individual cell mass, measured as dry weight, was constant over a range of fast growth rates (,u = 0.4 to 0.7) but increased at slower growth rates (Fig. 3A). It is conceivable that cell mass increased because large numbers of cells spontaneously became dipl6id at slow growth rates. This does not, however, appear to be the case since in cultures of the isogenic diploid strain cell mass is higher than that of the haploid even at slow growth rates (unpublished observations). The dry weight of individual cells probably increased because large quantities of storage materials were accumulated at slow growth rates. The RNA content of cells in different media was determined by measurements of ["4C luracil incorporation. The amount of RNA as a percentage of dry weight decreased linearly with decreasing growth rate between ,u = 0.7 and 0.2.

859

RNA COMPOSITION IN YEAST

VOL. 122, 1975

A

t

24

._1

*

a

a

2

:. 0

12

.

2

B 0.12

~0.0 3t

100.04 z 2

a 3

E

1.6iC z a

1.2 0

120 4i Turbidity (Klett units)

FIG. 2. Demonstration of balanced growth in yeast. Cultures were grown in parallel in medium F. (A) Culture dry weight; (B) protein content; (C) RNA content; (D) DNA content.

0-1

85 OA

X

I~~~~~~~~~~~~~~~~~~~~

z

This relationship did not hold at very slow growth rates since RNA constituted about the same fraction of dry weight when g = 0.05 as 0.2 04 0.6 Growth rate (generations h-1) when u 0.2 (Fig. 3B). The RNA content per cell also decreased with decreasing growth rate, FIG. 3. Effect of growth rate on dry weight and but in a manner that can best be represented by RNA content of yeast cells. (A) Dry weight per cell; the curvilinear function shown in Fig. 3C. These (B) the RNA fraction of dry weight; (C) RNA per cell. measurements of RNA content were not corrected for the DNA labeled since DNA was only TABLE 2. Nucleic acid content of yeast cells growing a small fraction of total nucleic acid (Table 2). in different media Although the method of DNA estimation used is not very accurate (-+8%; 30), the data do RNA (% dry wt) DNAa suggest that the amount of DNA is about 0.02 Growth pg/cell, independent of growth rate. Given that Medium rate d Hydro(GU) lytic cellular DNA content is constant, the RNA/ dr tpg/cell mto( ethoda DNA ratio will change with growth rate in the same manner as the RNA/cell ratio changes C 0.18 0.025 12.7 11.2 0.69 (Fig. 3C). Y 8.5 7.0 0.45 0.16 0.023 Published estimates of RNA content in yeast 8.7 F 0.38 0.11 0.016 5.8 vary widely (17, 26, 32). Some of this variation 5.5 3.4 P 0.24 0.09 0.018 L 4.9 2.6 is probably due to the use of unreliable methods 0.10 3.9 2.6 E 0.08 0.10 0.023 for RNA determinations. To demonstrate the validity of RNA estimations by isotope incorpoa Estimated from absorbance at 260 nm of alkaliration, we measured the RNA content of yeast resistant, deoxyribonuclease-sensitive cellular matecultures in different media by a completely rial. independent procedure. In these experiments b Estimated from absorbance at 260 nm of alkaliRNA was assayed by the absorbance of 260 nm labile cellular material. of acid-precipitable, alkali-soluble cellular ma"Estimated from [14C uracil incorporation; values terial (30). This hydrolytic assay gave slightly for these growth rates are taken from Fig. 3B. =

Isotopic

m

860

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WALDRON AND LACROUTE

higher values for RNA content than did the isotope incorporation procedure (Table 2), probably due to overestimation of RNA by the hydrolytic method. In this assay the absorbance at 260 nm could easily be increased by the presence of material other than the products of RNA solubilization. Nevertheless, the two methods give estimates for RNA of the same order of magnitude, and both show that RNA content is dependent on growth rate. RNA extraction is not affected by growth rate. It is essential for our analysis that RNA is extracted from cells growing in different media without selective loss of any type of RNA at any growth rate. To demonstrate that our preparations of extracted RNA are representative of cellular RNA, we compared the yields of stable and nascent RNAs from cells growing at different rates. If nascent RNA (rich in precursors and other species of unstable RNA) is extracted with the same efficiency as the stable RNA, then the extraction procedure can be regarded as nonselective. Cultures in media C, Y, P, or E were incubated overnight in the presence of [3H ]uracil. The cells from each culture were then collected on sterile filters and resuspended in the same medium but containing only nonradioactive uracil. The cells were incubated for about 5 h in nonradioactive medium to chase all the [3H ]uracil into long-lived RNA. Newly synthesized RNA in these cultures was labeled with [IC Icytosine for only 5 or 10 min, depending on growth rate. Cytosine was used for this shortterm labeling because it is taken up at a high rate even in the presence of the large amount of external uracil necessary to support growth of strain FL 521 (unpublished observation). After RNA extraction, the 3H/"4C ratios in cells and RNA preparations were determined by alkaline hydrolysis to render all incorporated counts soluble (2). The 3H/14C ratio was characteristic for each experiment, but with each medium the ratio in cellular RNA was the same as that in purified RNA (Table 3). Therefore our extraction procedure did not result in selective loss of stable or nascent RNAs from cells growing within the range of rates under investigation. Estimation of the amounts of rRNA and tRNA. Total RNA was analyzed by electrophoresis through slabs of 2.5% polyacrylamide gel strengthened with 0.5% agarose (20). By this procedure the RNA was separated into three major components (Fig. 4). The slowest-moving band consisted mainly of 25S rRNA. This band also contained the 5.8S rRNA, which was only released from the 25S RNA under strongly dissociating conditions (28; see also below). The second band in region A of

TABLE 3. Efficiencies of extraction of stable and nascent RNA from cells growing in different mediaa 3H/14C

Medium

In cells

In RNA

0.75 0.43 1.71 16.8

0.82 0.44 1.72 14.5

C Y

P Eb

a Stable RNA was labeled by overnight incubation with [3H]uracil (0.25 ACi/ml; 10 gg/ml), followed by a 5-h chase with nonradioactive uracil (10 ug/ml). Nascent RNA was labeled by 5 min of incubation with ["4C]cytosine (0.5 jsCi/ml; 3 tsg/ml). b Nascent RNA was labeled by 10 min of incubation with [14C]cytosine (0.5 uCi/ml; 3 ug/ml).

B

A

1a

II~~6 E a.

x

5

0

2

i

6

8

Distance migrated (cm)

FIG. 4. Separation of yeast RNA into large and small species. Total RNA was extracted from cells labeled with ['4C]uracil in medium C and subjected to electrophoresis through gels of 2.5% polyacrylamide. Migration was from left to right. Region A contains rRNA (25S and 5.8S in the large peak and 18S in the smaller peak). Region B contains tRNA and 5S rRNA.

the gels (Fig. 4) contained the 18S rRNA. Since these rRNAs had about the same pyrimidine content (7), the ratio of counts in the two peaks of region A should be the same as the ratio of their molecular weights, i.e., 1.35:0.7 or 1.93:1. The observed ratios were in good agreement with this figure, varying only between 1.85:1 and 1.97:1. Since the ratio was independent of growth rate, the rRNAs in region A must have

VOL. 122, 1975

RNA COMPOSITION IN YEAST

been present in equimolar amounts at all growth rates. Very rarely a preparation contained the two bands in a much lower ratio. Data from such preparations were not used since the low ratio indicated some degradation. In addition to mature rRNA, region A also contained some ribosomal precursor RNA (29) and some messenger RNA (mRNA). However, rRNA precursors are present only in very small amounts (29). mRNA of this size is also present in only small quantities since yeast mRNA is monocistronic (21) and only a minor fraction of yeast polypeptides is long enough to require such large messengers (31). We have therefore assumed for this study that the amounts of ribosomal precursor and mRNA in region A are

A 90

* 4

z 80 a

2 o0 70 B

negligible.

The fast-moving band in region B of these gels (Fig. 4) consisted of 4S tRNA and 5S rRNA. The 5S RNA was present in an equimolar ratio with the large rRNA species (see below). Total rRNA was therefore calculated as the RNA in region A together with the estimated amount of 5S RNA. tRNA was calculated as the amount of RNA in region B after subtraction of the 5S RNA. Effect of growth rate on the amounts of rRNA and tRNA. Total RNA from cells growing in different media was analyzed by electrophoresis as described above. At fast growth rates (.u = 0.7), rRNA constituted about 85% of total RNA. This fraction decreased slightly with decreasing growth rate (Fig. 5A). The proportion of tRNA, on the other hand, increased with decreasing growth rate (Fig. 5B). It follows that the tRNA/rRNA ratio increased with decreasing growth rate (Fig. 5C). From estimates of the tRNA fraction (Fig. 5B) and of total RNA content (Fig. 3C), we could calculate the number of tRNA molecules per cell in each growth medium (Table 4). We could likewise calculate the number of ribosomes per cell, assuming that all rRNA is present in ribosomes (Table 4). The number of tRNA molecules per ribosome in yeast was similar to that in both bacteria (27) and rat liver (23). Relative amounts of 4S and 5S RNA. The 5S RNA is present in equimolar amounts with the other rRNAs in mature ribosomal particles (28). To show that this is also true in preparations of total RNA (which would include pools of free RNA), we compared the ratios of 4S to 5S RNA in preparations with the'ratios predicted if all rRNAs' `.are present in `equ'al numbers of molecules.. Total RNA was subjected to electrophoresis through 'gels of 7.5%.'piolyacrylamide, which the large RNAs do -not enter.-Little'or no 5.8S.rRNA.was found at the.expected position.

861

20B

10

C

0.2

tRNA r RNA

"

0.1

_

_._

_

OA 0.2 0.6 Growth rate (generation I

h)

FIG. 5. Effect of growth rate on the fraction (by weight) of rRNA and tRNA. (A) rRNA as a percentage of total RNA; (B) tRNA as a percentage of total RNA; (C) tRNA/rRNA ratio (by weight). Best-fitting lines were calculated by regression analysis in these gels (Fig. 6), and this RNA must therefore be complexed with the 25S RNA (28). The 4S and 5S RNAs were present in the gels in clearly, separated bands (Fig. 6). At all growth rates. the 4SI5S ratio was 'similar to that predicted. on the assumption that all rRNAs are present in. equimolar amounts (Fig. 7). Thus the corrections made. for'6S.RNA 'present in region B-',of 2'.5% polyacrylamide. gels were valid (see above). Furthermore, the.decrease in'the 4S/5S ratio as; growth rate increased (Fig.: 6 and 7) .confir.ms':that rRNA and tRNA are not maintained in the'same' ratio at all growth rates. Ribosome. efficiency at different growth

862

WALDRON AND LACROUTE

TABLE 4. Number of tRNA molecules and ribosomes in yeast cells growing in different media Growth Medium

ratea

C Y F P L E

0.62 0.46 0.41 0.25 0.076 0.056

No. of No. of No. of tRNA moleribosomes tRNA molecules per per cell cules per cell (x 106) (x 105)O ribosome

3.30 2.57 2.33 1.78 1.76 1.82

3.48 2.52 2.23 1.58 1.46 1.49

9.5 10.2 10.4 11.2 12.0 12.2

Average values. bAssuming an average molecular weight for yeast tRNA of 2.5 x 104. c Assuming that each ribosome contains 2.09 x 106 daltons of rRNA and all rRNA is present in ribosomes. a

J. BACTERIOL.

RNA polymerases are present in about the same amounts as in growing cells (10). From studies of this situation, it has been postulated (10) that yeast RNA synthesis is controlled by the fraction of polymerase molecules active in RNA synthesis and by the rate at which they transcribe the DNA template. Such regulation of polymerase activity could be mediated by factors like the protein 7r, which was isolated from yeast cells and which can stimulate the in vitro activity of yeast RNA polymerases (6). The poorly studied phenomenon of RNA degradation may also be important in determining the amount of RNA in cells growing at steady state.

rates. During steady-state growth, the amount of any cell component will be doubled in one generation time. Therefore, the net rate of protein synthesis can be calculated from the protein content at steady state and the growth rate of the culture (16). We measured these two parameters for yeast cells growing in different media and thereby calculated the net rate of protein synthesis at different growth rates (Table 5). We could also estimate cellular ribosome content at these growth rates from the data already presented. It was thus possible to show that ribosome efficiency, expressed as the rate of protein synthesis per ribosome, decreased with decreasing growth rate (Fig. 8).

DISCUSSION We have measured the RNA content of yeast cells by two completely different methods. Both procedures give similar values, which are intermediate in the range of published estimates (26, 32). We found that the total RNA content of yeast cells decreases with decreasing growth rate in a nonlinear fashion. Previous reports have described linear decreases in RNA content as a function of growth rate in yeast (17, 26). However, our observations were made on batch cultures and do not necessarily contradict the other findings, which were made with chemostat cultures. There are a number of mechanisms by which the changes in RNA content could be achieved. It has been suggested that the number of RNA polymerase molecules is a major factor in controlling the RNA content of chemostat-grown yeast cells (26). However, the activity of polymerase molecules, as well as their number, can be regulated. For example, there is little RNA synthesis in glucose-starved or cycloheximide-inhibited yeast cells, yet the

E

a. Q x

O

0

2

4

6

8

Distance migrated (cm) FIG. 6. Electrophoretic separation of 4S and 5S RNAs. Total RNA was extracted from cells grown in the presence of [14C]uracil. The 4S and 5S RNA species were separated by electrophoresis of the RNA through 7.5% polyacrylamide gels. Migration is from left to right. The 4S RNA has migrated about 7 cm and the 5S RNA about 5.3 cm. Any 5.8S RNA would have migrated about 4 cm. (A) RNA from cells grown in medium E; (B) RNA from cells grown in medium C.

VOL. 122, 1975

8

*. 6

4S 5S 4

2

0.2

Growth

0.4 rate

0.6

(generation/k)

FIG. 7. Ratio (by weight) of 4S to 5S RNAs after separation in 7.5% polyacrylamide gels. The predicted ratios (-----) were calculated from the relative amounts of rRNA and tRNA (Fig. 5C), assuming that all rRNA's are present in equimolar amounts and hence 5S RNA constitutes 1.9% (by weight) of total rRNA. TABLE 5. Rate of protein synthesis in cells growing in different media Net rate

Growth

Medium

rate

(,)

Protein content

(pg/cell)

of protein

synthesis

(1O-1 amino acids/s per cell)"

C Y F P L E

863

RNA COMPOSITION IN YEAST

0.59 0.48 0.42 0.21 0.08 0.04

3.04 2.76 1.98 1.62 2.54 1.90

19.4 14.3 9.0 3.7 2.1 0.7

existence of two such polymerases has not yet been demonstrated in yeast but is suggested by the fact that the structural genes for tRNA are not linked to the clustered rRNA cistrons (25). A polymerase specific for tRNA has, however, been identified in mammalian cells and is distinct from the nucleolar polymerase that synthesizes rRNA (34). The increase in rRNA/ tRNA ratio at fast growth rates may be due to an increase in the fraction of rRNA that is present in ribosomal precursors. In this case the number of tRNA molecules per functional ribosome may be the same at all growth rates. We have observed that yeast 5S rRNA is present in equimolar amounts with the other rRNA species at all growth rates. The 5S rRNA is not synthesized in the same precursor molecule as the other rRNAs (29), but the cistrons for 5S RNA are interspersed with those for the other rRNAs (25). Thus coordination of the amounts of different rRNAs in yeast could be achieved by one polymerase synthesizing all of the species. This is not the case in higher organisms, where the genes for 5S RNA are not linked to other rRNA cistrons (25) and 5S RNA is produced by a distinct, extranucleolar polymerase (34). A decrease in growth rate causes a reduction in the net rate of protein synthesis in yeast. Surprisingly, this reduction does not appear to be due to limiting numbers of ribosomes. In fact, the rate of protein synthesis per ribosome (ribosome efficiency) decreases quite markedly with decreasing growth rate. The number of tRNA molecules does not seem to be the limiting factor either, since the number of tRNAs per ribosome actually appears to increase slightly with decreasing growth rate. It is possible that

a Calculated as: (protein per cell x Avogadro's number x In 2)/107 x generation time (s), assuming that the average molecular weight of amino acids in yeast protein is 107 (32).

6

Growth rate affects not only the quantity of RNA in yeast cells but also its composition. With decreasing growth rate the amounts per cell of both rRNA and tRNA are reduced, but E by different extents. Hence, as the growth rate decreases the ribosomal fraction of RNA decreases slightly whereas the tRNA fraction increases. These are the same trends observed in other microorganisms, both prokaryote and euI I karyote (3, 14; Plaut and Turnock, personal 04 02 06 Growth communication). The non-coordination of (gen9rations/h) rRNA and tRNA levels may be due to controls FIG. 8. Ribosome efficiency as a function of growth operating on distinct polymerases, one of which rate. The efficiency was calculated as the rate of synthesizes rRNA and the other tRNA. The protein synthesis per ribosome.

l1

rate

864

WALDRON AND LACROUTE

J. BACTERIOL.

protein synthesis is limited by the cytoplasmic 8. Forchhammer, J., and N. 0. Kjeldgaard. 1968. Regulation of messenger RNA synthesis in Escherichia coli. J. concentration of either ribosomes or tRNA molMol. Biol. 37:245-255. ecules. Unfortunately, this problem cannot 9. Forchhammer, J., and L. Lindahl. 1971. Growth rate of readily be studied in yeast cells because they polypeptide chains as a function of cell growth rate in a contain vacuoles and therefore measurements of mutant of Escherichia coli 15. J. Mol. Biol. 55:563-568. cytoplasmic volume are extremely difficult. The 10. Gross, K. J., and A. 0. Pogo. 1974. Control mechanism of ribonucleic acid synthesis in eukaryotes. The effect of finding that ribosome efficiency in yeast deaminoacid and glucose starvation and cycloheximide creases with growth rate is in sharp contrast to inhibition on yeast deoxyribonucleic acid-dependent the situation in bacteria, where ribosomes are ribonucleic acid polymerases. J. Biol. Chem. 249:568-576. used with maximum efficiency over a wide L. H. 1967. Macromolecular synthesis in temrange of growth rates (3, 9, 16). However, a 11. Hartwell, perature-sensitive mutants of yeast. J. Bacteriol. dependency of ribosome efficiency on growth 93:1662-1670. rate has also been deduced from studies of T. 12. Hindley, J., and S. M. Page. 1972. Nucleotide sequence of yeast 5S ribosomal RNA. FEBS Lett. 26:157-160. pyriformis (14), Prototheca zopfii (22), and A. L. 1971. The adaptive responses of Escherichia Physarum polycephalum (Plaut and Tumock, 13. Koch, coli to a feast and famine existence. Adv. Microb. personal communication). Thus it may be a Physiol. 6:147-217. general phenomenon that in eukaryotic micro- 14. Leick, V. 1967. Growth rate dependency of protein and nucleic acid composition of Tetrahymena pyriformis organisms ribosomes are used with very high and the control of synthesis of ribosomal and transfer efficiency only at fast growth rates. On the other RNA. C.R. Trav. Lab. Carlsberg 36:113-126. hand, it must be emphasized that there is no 15. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. information available about protein stability in Randall. 1951. Protein determination with the Folin phenol reagent. J. Biol. Chem. 193:265-275. these organisms. If protein turnover does occur, O., and N. 0. Kjeldgaard. 1966. Control of ribosome efficiencies will have been underesti- 16. Maaloe, macromolecular synthesis. W. A. Benjamin Inc., New mated because they were calculated from the York. net rates of protein synthesis and not the total 17. McMurrough, I., and A. H. Rose. 1967. Effect of growth rates. ACKNOWLEDGMENTS We are very grateful to Barbara Plaut and Geoffrey Turnock for sending us their data on Physarum polycephalum before publication and for emphasizing the possible importance of protein turnover. Thanks are also given to MarieLouise Bach for providing strain FL 521 and for reading the manuscript. Our work was supported by a postdoctoral fellowship of the Royal Society European Exchange programme to C.W. and a gift from the French Fondation pour la Recherche Medicale. LITERATURE CITED 1. Beaven, G. H., E. R. Holiday, and E. A. Johnson. 1955. Optical properties of nucleic acids and their components, p. 493-553. In E. Chargaff and J. N. Davidson (ed.), The nucleic acids, vol. 2. Academic Press Inc., New York. 2. Dennis, P. P., and H. Bremer. 1974. Differential rate of ribosomal protein synthesis in Escherichia coli B/r. J.

Mol. Biol. 84:407-422. 3. Dennis, P. P., and H. Bremer. 1974. Macromolecular composition during steady-state growth of Escherichia coli B/r. J. Bacteriol. 119:270-281. 4. Dennis, P. P., and R. K. Herman. 1970. Control of deoxyribonucleic acid and ribonucleic acid synthesis in pyrimidine-limited Escherichia coli. J. Bacteriol. 102:124-129. 5. De Wachter, R., and W. Fiers. 1971. Fractionation of RNA by electrophoresis in polyacrylamide gel slabs. Methods Enzymol. 21:167-178. 6. Di Mauro, E., C. P. Hollenberg, and B. Hall. 1972. Transcription in yeast: a factor that stimulates yeast RNA polymerases. Proc. Natl. Acad. Sci. U.S.A. 69:2818-2822.

7. Fauman, M., M. Rabinowitz, and G. S. Getz. 1969. Base composition and sedimentation properties of mitochondrial RNA of Saccharomyces cerevisiae. Biochim. Biophys. Acta 182:355-360.

rate and substrate limitation on the composition and structure of the cell wall of Saccharomyces cerevisiae. Biochem. J. 105:189-203. 18. Mounolou, J. C. 1971. The properties and composition of yeast nucleic acids, p. 309-333. In A. H. Rose and J. S. Harrison (ed.), The yeasts, vol. 2. Academic Press Inc., New York. 19. Osawa, 0. 1960. The nucleotide composition of ribonucleic acids from subcellular components of yeast, Escherichia coli and rat liver, with special reference to the occurrence of pseudouridylic acid in soluble ribonucleic acid. Biochim. Biophys. Acta 42:244-254. 20. Peacock, A. C., and C. W. Dingman. 1968. Molecular weight estimation and separation of ribonucleic acid by electrophoresis in agarose-acrylamide composite gels. Biochemistry 7:668-674. 21. Petersen, N. S., and C. S. McLaughlin. 1973. Monocistronic messenger RNA in yeast. J. Mol. Biol. 81:33-45. 22. Poyton, R. 0. 1973. Effect of growth rate on the macromolecular composition of Prototheca zopfii, a colorless alga which divides by multiple fission. J. Bacteriol. 113:203-211. 23. Quincey, R. V., and S. H. Wilson. 1969. The utilization of genes for ribosomal RNA, 5S RNA and transfer RNA in liver cells of adult rats. Proc. Natl. Acad. Sci. U.S.A. 64:981-988. 24. Rubin, G. M. 1973. The nucleotide sequence of Saccharomyces cerevisiae 5.8S ribosomal ribonucleic acid. J. Biol. Chem. 248:3860-3875. 25. Rubin, G. M., and J. E. Sulston. 1973. Physical linkage of the 5S cistrons to the 18S and 28S ribosomal RNA cistrons in Saccharomyces cerevisiae. J. Mol. Biol. 79:521-530. 26. Sebastian, J., F. Mian, and H. 0. Halvorson. 1973. Effect of the growth rate on the level of the DNA-dependent RNA polymerases in Saccharomyces cerevisiae. FEBS Lett. 34:159-162. 27. Skjold, A. C., H. Juarez, and C. Hedgcoth. 1973. Relationships between deoxyribonucleic acid, ribonucleic acid, and specific transfer ribonucleic acids in Escherichia coli 15T- at various growth rates. J. Bacteriol. 115:177-187.

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28. Udem, S. A., K. Kaufman, and J. R. Warner. 1971. Small ribosomal ribonucleic acid species of Saccharomyces cerevisiae. J. Bacteriol. 106:101-106. 29. Udem, S. A., and J. R. Warner. 1972. Ribosomal RNA synthesis in Saccharomyces cereuisiae. J. Mol. Biol. 65:227-242. 30. Von Meyenburg, K. 1971. Transport-limited growth rates in a mutant of Escherichia coli. J. Bacteriol. 107:878-888. 31. Waldron, C., R. Jund, and F. Lacroute. 1974. The elongation rate of proteins of different molecular weight

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classes in yeast. FEBS Lett. 46:11-16. 32. Wehr, C. T., and L. W. Parks. 1969, Macromolecular synthesis in Saccharomyces cerevisiae in different growth media. J. Bacteriol. 98:458-466. 33. Weil, J. H. 1969. Distribution subcellulaire des divers tRNA de levure et d'E. coli. Bull. Soc. Chim. Biol. (Paris) 51:1479-1496. 34. Weinmann, R., and R. G. Roeder. 1974. Role of DNAdependent RNA polymerase m in the transcription of the tRNA and 5S RNA genes. Proc. Natl. Acad. Sci. U.S.A. 71:1790-1794.