GROSS A N D POGO
Scatchard, G. (1949), Ann. N.Y. Acad. Sci. 51 660. Sidman, J. W. (1956), J. Am. Chem. SOC.78, 4561. Smith, B. (1969), Br. Heart J. 31, 607. Theologides, A., Yarbro, J. W., and Kennedy, B. J. (1968), Cancer 21, 16. Ward, D. C., Reich, E., and Goldberg, I. H. (1965), Science ~
149, 1259. Waring, M. J. (1970), J . Mol. Biol. 54, 247. Yamamoto, K., Acton, E. M., and Henry, D. W. (1972), J . Med. Chem. 15. 872. Zunino, F., DiMarco, A., Zaccara, A., and Juoni, G. (1974), Chem.-Biol. Interact. 9, 25.
Control of Ribonucleic Acid Synthesis in Eukaryotes. 2. The Effect of Protein Synthesis on the Activities of Nuclear and Total DNA-Dependent RNA Polymerase in Yeast+ Kurt J. Gross and A. Oscar Pogo*
ABSTRACT: A thermosensitive conditional yeast mutant
(ts-l87) which suppresses protein synthesis at the nonpermissive temperature (36 "C) also suppresses RNA synthesis. The effect of temperature on the mutant is similar to the addition of cycloheximide-it inhibits the incorporation of labeled precursors into RNA in both whole cells and isolated nuclei. The effect of temperature is selective for the RNA polymerases bound to the nuclear template but not for the total R N A polymerases. Thus, the specific activities and total amounts of R N A polymerase species extracted and assayed with exogenous DNA template are similar in the t,-187 cultured at 23 "C and at 36 OC. On the contrary, the nuclear polymerases, Le., RNA synthesis in isolated nuclei, are dramatically inhibited in cells cultured at 36 OC.When amino acid starved t,-187 cells are transferred to 36 OC,release from the inhibition of RNA synthesis is observed. As with the addition of cycloheximide, this relaxation is observed in cells but not in isolated nuclei. The parental strain, A364A, which responds by stimulating instead of inhibiting protein synthesis when the temperature is increased to 36 OC, also exhibits an inhibition in the incorporation of labeled precursor into R N A as well as reducing RNA synthesis in isolated nuclei. However, these are transitory inhibitions and afterward there is reinitiation of both processes. Reinitiation of R N A synthesis in isolated nuclei is
s t r i n g e n c y has been defined as a gene-controlled process that exists in prokaryotes by which they can adapt to environmental changes of nutrients (Edlin and Broda, 1968). It operates by inhibiting the RNA synthetic machinery as well as the protein synthetic machinery in cells cultured in an amino acid deprived medium (Ryan and Borek, 1971). Although the coupling of both machineries had been originally observed in eukaryotes (Brachet, 1957), it is currently accepted that stringency is a genetic phenomenon restricted to prokaryotes (Edlin and From the Laboratory of Cell Biology, The Kimball Research Institute of The New York Blood Center, New York, New York 10021. Receiued October 17,1975. This work was supported by Grant HL-09011 from the National Heart and Lung Institute and Grant CA17626 from the National Cancer Institute. This is the second paper in a series on control of RNA synthesis. The first one was Gross and Pogo (1974).
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similar to the relaxed phenomenon and it is called "nuclear relaxation". This relaxation can only be obtained if protein synthesis is not inhibited; however, cellular relaxation occurs in the absence of protein synthesis. The repression of the nuclear R N A polymerase activities which starvation and inhibition of protein synthesis produce appears to be due to a restriction in the nuclear DNA template. This notion is supported by the fact that a net diminution of these nuclear enzyme activities is observed in spheroplasts cultured under starving conditions. Studies of the four main ribonucleotide pools indicate that stringency and inhibition of protein synthesis (ts187 cultured at 36 "C) produce an increase in UTP and CTP pools. This is consistent with the concept that stringency and inhibition of protein synthesis affect the rate of utilization rather than the synthesis of these ribonucleotide residues. In the A364A and ts- 187 yeast strains, the conversion of uracil but not of uridine into the UTP and CTP is inhibited when there is inhibition of the nuclear RNA polymerases. This indicates that the uracil phosphoribosyltransferase but not the uridine-cytidine kinase is allosterically inhibited by UTP and C T P in yeast. The feedback inhibition in the metabolic pathway of the base explains why relaxation cannot be detected when uracil instead of uridine is used as the labeled RNA precursor.
Broda, 1968). Nevertheless, recent observations in ascites cells and yeast (Franze-Fern6ndez and Pogo, 1971; Gross and Pogo, 1974) clearly indicate that stringency is a universal procedure for regulating R N A metabolism. This implies that any biological process which produces stimulation or inhibition in the eukaryotic R N A synthetic machinery has to exert its effect through the function of RCstrgene(s) (Tsukada and Lieberman, 1965; Pogo et al., 1966, 1967; Doly et al., 1965; Tata, 1966; Rubin and Cooper, 1965; Pogo, 1972). The uncoupling of bacterial RNA and protein synthesis, known as relaxation, is the essential point of stringency since this phenomenon can be obtained by mutation (Stent and Brenner, 1961). The observation of phenotypic relaxation in yeast (Foury and Goffeau, 1973;Gross and Pogo, 1974) clearly established a further link in the basic mechanism by which
CONTROL O F RIBONUCLEIC ACID S Y N T H E S I S IN EUKARYOTES
RNA metabolism is modulated in eukaryotes and prokaryotes. At the present time, however, extensive studies in prokaryotes have not provided satisfactory explanation of the stringent and relaxed phenomena at the molecular level (Travers et al., 1970; Chen et al., 1972). Our studies in yeast indicate that these phenomena are mediated by the activities of at least two distinct factors having opposite effects on R N A synthesis-one functions by switching on R N A synthesis and the other by switching it off. The synthesis or activity of these two factors is under the influence of the protein synthetic machinery (Gross and Pogo, 1976). Materials and Methods Solutions for Enzyme Isolation from Total Spheroplasts. Buffer A contains 50 mM piperazine, N,N’-bis(2-ethanesulfonic acid), pH 7.8, 1 mM EDTA,’ 1 mM dithioerythritol (added just before use), 1 mM phenylmethanesulfonyl fluoride, 8 mM MgC12, 30% glycerol (v/v), and 0.3 mg/ml each of Dextran T70, T150, and T250 extensively dialyzed against distilled water. Buffer B which is the chromatographic buffer contains 50 mM piperazine, N,N’-bis(2-ethanesulfonicacid), pH 7.8, 0.1 mM EDTA, 0.5 mM dithioerythritol, 1 mM phenylmethanesulfonyl fluoride, 30% glycerol (v/v), and 0.3 mg/ml each of Dextran T70, T150, and T250. These buffers are treated with 1% Chelex-100 (w/v) (sodium form; Bio-Rad Laboratories) in order to remove trace amounts of heavy metals. Solutions for Nuclei Isolation. Buffer C contains 0.4 M sucrose, 10 mM piperazine, N,N’-bis(2-ethanesulfonic acid), pH 7.4, 10 mM NaCl, 5 mM MgC12, 1.6 mM MnC12, 1 mM dithioerythritol, and 0.01% spermidine. Solutionsfor Enzyme Isolation from Nuclei. Buffers D and E are equal to buffers A and B, respectively, but with 1 mg each of Dextran T70, T150, and T250. Yeast (Saccharomycesceriuisiae),Media, and Cell Growth. The haploid strains A364A and t,-187 (gall adel ade2 ural his7 lys2 t y r l ) were kindly supplied by Dr. L. Hartwell (University of Washington, Seattle). The haploid strain S288C (mal2 and gal2) was kindly provided by Dr. F. Sherman (University of Rochester). They were grown overnight to a final density of 2-3 X lo7 cells/ml in a complete medium containing (per liter) 6.7 g of yeast nitrogen base, 10 g of succinic acid, 6 g of NaOH, 20 g of glucose, 20 mg each of adenine and uracil, and 40 mg each of histidine, lysine, and tyrosine, adjusted to a final pH of 5.8.Cells were harvested by filtration onto Millipore filters, washed with distilled water, resuspended in fresh complete medium supplemented with streptomycin (50 Mg/ml) and penicillin (50 units/ml), and cultured for an additional hour before beginning the experiments. Metabolically active spheroplasts were prepared according to the procedure of Hutchinson and Hartwell (1967). After harvesting the cells by filtration as above, they were resuspended in 1 M D-sorbitol at a concentration of 2 X lo8 cells/ml. A commercial preparation of snail gut enzyme (Glusulase, Endo Laboratories) was added */loathvolume, and after 15 rnin of incubation at 23 OC for A364A and t,-187 and 30 rnin for S288C, the spheroplasts were harvested, washed once with 1 M D-sorbitol, and resuspended at concentrations (as explained in figures and tables) in complete medium but supplemented with 1 M D-sorbitol to provide osmotic support.
’
Abbreviations used are: UTP, uridine triphosphate; CTP, cytidine triphosphate; EDTA, ethylenediaminetetraacetic acid; DEAE, diethylaminoethyl; mRNA, messenger ribonucleic acid; tRNA, transfer ribonucleic acid.
Streptomycin (50 pg/ml) and penicillin (50 units/ml) were added to the medium and to all subsequent spheroplast cultures to prevent bacterial growth. To permit recovery from Glusulase treatment the spheroplasts were incubated under these conditions for 1 h before beginning the experiments. Nuclear Isolation. The main contaminants of the yeast nuclear fraction are cell wall fragments and intact cells. The former are very difficult to avoid, but the presence of intact cells is directly related to the efficiency by which the Glusulase treatment converts cells into spheroplasts. Thus, the A364A strain which we use is the one with the highest efficiency and the S288C is the one with the lowest efficiency in spheroplast formation. Spheroplasts were collected by centrifugation as described above, washed once with 1 M Dsorbitol, resuspended in buffer C at a proportion of 1:lO (spheroplast weight to buffer volume), and kept at -20 OC overnight. Triton X-100 and saponin were added to the spheroplast suspension to make a final concentration of 0.5% for each. Afterward, the suspension of detergent-treated spheroplasts was homogenized with a Teflon homogenizer (20 strokes at high speed) and centrifuged for 10 min at 10 OOOg. The supernatant was decanted, and the sediment was resuspended with one-half the original volume of buffer C minus detergents and homogenized again with 10 strokes at the same speed. Then it was centrifuged for 2 min at 8OOg to sediment cellular debris and cells. The supernatant was centrifuged at 10 OOOg for 10 rnin and the nuclear pellet was resuspended with 4-6 strokes of a Dounce homogenizer in one-half the volume of the same buffer and again centrifuged for 2 min at 8OOg to sediment any remaining contaminants. The final supernatant contained nuclei, insignificant amounts of cells in t,-187, and no cells in A364A. S288C spheroplasts were resuspended in buffer C, but containing 9% Ficoll (Pharmacia Fine Chemicals), lysed with the same detergents, and homogenized as above. The homogenate was centrifuged at lOOOg for 5 min and the supernatant centrifuged at 20 OOOg for 20 min. The sediment which contained the majority of nuclei was resuspended with a Dounce homogenizer in buffer C, but without Ficoll, and centrifuged at 8OOg for 2 min to sediment contaminants. The supernatant was then centrifuged at 10 OOOg for 12 min. The final sediment was resuspended with the Dounce homogenizer in the same buffer and centrifuged at 8OOg for 2 min to purify the nqclear suspension from rimaining cellular contaminants. Nevertheless, the final nuclear suspension contained 10-30% intact cells. Isolation of Total RNA Polymerase (RNA Nucleotidyltransferase, EC2.7.7.6).At the end of the corresponding periods of incubation, the spheroplasts were collected by centrifugation at 4000g for 7 rnin and washed once with 1 M Dsorbitol. The sedimented spheroplasts were resuspended at a ratio of 1:6 (w/v) in buffer A. This suspension was stored overnight in liquid nitrogen or processed immediately. Twelve-milliliter aliquots of the spheroplast suspension were transferred to a Rosett cell (Branson) kept at -10 OC in an ice-salt bath and sonicated with a Branson sonifer (Model W140D) using the microtip at a meter setting of 60. Total sonication time was 150 s, broken down into 15-s periods of sonication with 454 intervals. Disruption (of spheroplasts) was better than 90% when examined under a microscope, and the total homogenate was centrifuged in a Spinco 65 rotor at 42 000 rpm for 60 min. The high-speed supernatant was stored in liquid nitrogen for not longer than 48 h or immediately subjected to DEAE-Sephadex chromatographic fractionation. Isolation of A364A Nuclear RNA Polymerase. This is the only strain which yielded a nuclear fraction practically uncontaminated by cells. The presence of cells was determined BIOCHEMISTRY, VOL.
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by dissolving the nuclei with addition of sodium dodecyl sulfate (1%) to the nuclear suspension and examining the lysate under the microscope. The nuclei were prepared as described above, in buffer C, and centrifuged for 10 min at 10 OOOg. The sediment of packed nuclei was resuspended in buffer D at a concentration of approximately 200 pg of DNA/ml. To this suspension was added dropwise an equal volume of buffer D but with the addition of 2 M ammonium sulfate. The viscous nuclear suspension was sonicated with the Branson sonifer using the microtip at a meter setting of 35. Total sonication was 5 s/ml, broken down into 10-s periods of sonication with 30-s intervals. The suspension was then centrifuged in a Spinco 65 rotor at 42 000 rpm for 60 min. This high-speed supernatant was assayed and found to be totally dependent on exogenous DNA template. It was then subjected to ammonium sulfate precipitation in order to recover the nuclear enzymes in a solution without ammonium sulfate. The l M ammonium sulfate supernatant was made up to 60% saturation by gradual addition of a fine powder of ammonium sulfate added at a rate of 0.4 g/5 rnin and the solution was allowed to mix in the cold for an additional 30 min to ensure complete precipitation of the RNA polymerase activity. The preparation was centrifuged in the Spinco 65 rotor at 45 000 rpm for 60 rnin and the sediment was resuspended in buffer D at half the volume of the starting material. Finally, it was subjected to DEAE-Sephadex chromatographic fractionation, but in order to further reduce the starting ammonium sulfate concentration the preparation was diluted in an equal volume of buffer D immediately prior to use. DEAE-Sephadex Chromatography. For the fractionation of total enzymes 5 mg of protein of the high-speed supernatant and for the extracted nuclear enzyme an aliquot equivalent to 1.2 X 1O'O nuclei with a specific activity of 160 pmol UMP/lOs nuclei was loaded onto a DEAE-Sephadex A-25 column (1 .O X 15 cm) equilibrated with 0.03 M ammonium sulfate in the respective chromatographic buffers B and E. After loading, the column was washed with one column volume of 0.03 M ammonium sulfate in chromatography buffer and one column volume of 0.06 M ammonium sulfate in chromatography buffer, and then eluted with six column volumes of a linear gradient 0.06-0.35 M ammonium sulfate. A flow rate of 15 ml/h was used; 1.5-ml fractions were collected, and 50 p1 was assayed in a final incubation volume of 165 p1 containing the components mentioned above and 20 pg of native salmon sperm DNA. Incubation and counting conditions have already been explained (see below and Gross and Pogo, 1974). To determine total activity the enzyme fractions were pooled and RNA polymerases were assayed at saturating substrate concentrations (see respective tables). A 70-90% enzyme recovery was obtained with these chromatographic fractionation procedures. For this calculation, the activity of the high-speed supernatant assayed in the presence of 0.1 M ammonium sulfate and denatured DNA was taken as representative of the total input activity. All enzyme species, before and after fractionation on DEAE-Sephadex, were completely dependent upon the addition of DNA template. Assay of Isolated Nuclear and Total RNA Polymerase. The condition of enzymatic assays was similar to those described (Gross and Pogo, 1974). The assay mixture was incubated at 23 OC for 10 min and the reaction was stopped as explained (Gross and Pogo, 1974). The value of 2.0 pg of DNA/ lo8 cells corrected for mtDNA (-1 5%) (Grimes et al., 1974) for haploid cells was used to calculate enzymatic activities per cells. Assay of RNA Synthesis in Isolated Nuclei. The condition
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of enzymatic assay of isolated nuclei was similar to those described (Gross and Pogo, 1974). The assay mixture was incubated at 23 OC for 30 min and the reaction was stopped as explained (Gross and Pogo, 1974). The value of 2.0 pg of DNA/ lo8 nuclei corrected for mtDNA (-1 5%) was used to calculate nuclear enzymatic activities per nuclei. RNA and Protein Synthekis Measurements. [5-3H]Uridine (29 Ci/mmol) or [5,6-3H]uracil (44 Ci/mmol) and L[3H]ly~ine(7 Ci/mmol; Schwarz/Mann) was added as explained in the corresponding figures. Incorporation of the radioactivity was determined as follows: duplicate aliquots were precipitated in 10% cold trichloroacetic acid and collected on Whatman GF/C filters. The filters were washed three times with cold 5% trichloroacetic acid and once with ethanol, dried, and placed in vials. Triton-toluene scintillation mixture was added and the radioactivities were counted in the Beckman liquid scintillation counter. All radioactivity was detected in the RNA moiety. Incorporation of ~ - [ ~ H ] l y s i nwas e determined as follows: duplicate aliquots were precipitated in 10% trichloroacetic acid, heated to 90 OC for 15 min, and cooled; then the hot acid-insolublematerial was collected on Whatman G F / C filters and processed as explained above. Determination of Ribonucleotide Pools and Rates of Uracil Conversion into UTP and CTP and UMP Incorporation into Total RNA. A modification of the method of Winslow and Lazzarini (1969) was used to study total amount of cellular ATP, CTP, GTP, and UTP, uracil conversion into UTP and CTP, and UMP incorporation into total RNA. The cells were cultured overnight in complete medium supplemented with H332P04 (specific activity 30 mCi/mmol). They were then harvested by filtration onto Millipore filters, resuspended in fresh complete medium with an identical H332P04 specific activity, and cultured for an additional 50 min. They were collected by filtration, resuspended in fresh uracil-deprived medium with the same specific activity of H332P04, and cultured for 10 min. To determine any changes in the H332P04 specific activity in the medium, samples were withdrawn prior to incubation in the uracil-deprived medium and at the end of the 10-min culture. At this time [5,6-3H]uracil was added as explained in Figure 5 and 1-ml samples were withdrawn and mixed with 0.5 ml of ice-cold 4 M formic acid. The acid-treated cells were allowed to settle in the cold for 30 rnin and centrifuged at 1200g for 15 min. The supernatant was decanted and mixed with 5 pl of a solution containing 10 pM each of ATP, GTP, CTP, and UTP. To each sample 0.25 ml of a Darco G-60 charcoal suspension (4% w/v) in 1 mM HCI was added, and the mixture was incubated in the cold for 15 min. The charcoal was removed by centrifugation, washed once with 1.25 ml of 1 mM HCl in the cold, and resuspended in 1 ml of ethanolammonium hydroxide-water (66:0.3:33, v/v). This suspension was transferred to capillary pipets plugged with glass wool, 0.5 cm of washed Celite to serve as a filter, and washed with 1 ml of the ethanol-ammonium hydroxide-water mixture. The effluent and wash were collected in scintillation vials, concentrated to approximately 0.25 ml, and taken to dryness over KOH in a vacuum desiccator. The dry material was dissolved in 50 p1 of water and 2 0 4 samples were chromatographed on PEI-cellulose plates as explained (Winslow and Lazzarini, 1969). The UMP incorporation into total RNA was done in the presence of H332P04 as explained before and by the addition of [5,6-3H]uracil as explained in the legend of Figure 6. Four-milliliter samples were withdrawn at the indicated intervals and mixed with equal volumes of cold 10% trichloroacetic acid containing 1 mg/ml of nonradioactive uracil. The cells were collected by centrifugation and washed three times
CONTROL OF RIBONUCLEIC ACID SYNTHESIS IN EUKARYOTES
A364A ''Or
6or
I
ts-187
I
r
/
u
;8.0 H P V w
f
-
60-
30
40-
20 -
+Ty36"
,-o
5
Lo
5
n
4.0
I
I
R1
U m
IO -
20 -
20
40
60
2.0 20
40
60
MINUTES
FIGURE 1: Incorporation of L-[3H]ly~inein A364A (a, left) and t,-187 (b, right) cells during temperature shift from 23 to 36 OC.The cells were resuspended in 60 ml of medium at a density of 2 X lo7 cells/ml; after 30 rnin of incubation at 23 OC, 150 pCi of lysine was added to each and 30 rnin later 30 ml of each culture was transferred to a 36 OC prewarmed flask. Duplicate aliquots (2.5 ml) were taken at the indicated intervals and radioactivities measured as explained (Materials and Methods). Arrow, time of temperature shift; 0-0,23 OC;A---A, 36 O C .
with 10 ml of 2.5% cold trichloroacetic acid containing the same amount of nonradioactive uracil. The efficiency of washing was monitored by determination of radioactivity in the last wash. The sedimented cells were resuspended in 0.1 ml of distilled water and 0.8 ml of 0.3 M KOH was added. The alkaline hydrolysate was processed and [5,6-3H]- and [32P]U M P were determined after chromatographic fractionation on PEI-cellulose as explained (Winslow and Lazzarini, 1969) Protein and DNA Measurements. Protein was determined according to the method of Crampton (Crampton et al., 1954) and DNA by Burton's procedure (Burton, 1953). Results Effect of Temperature Shift During Lysine Incorporation in the A364A and t,-187. The effect of an increase of temperature from 23O to 36 OC on ~ - [ ~ H ] l y s i incorporation ne in the parental A364A and the thermosensitive t-187 mutant is presented in Figure 1. After the shift, an increase in lysine uptake in the A364A is produced (Figure la), but is suppressed in about 8-10 rnin in the t,-187. The inhibition of protein lynthesis in the t,-187 at 36 OC is expected since at this temperature there is a rapid breakdown of polysomes (Hartwell and McLaughlin, 1969), a disappearance of cytoplasmic mRNA, and a blockage of mRNA transport with accumulation of mRNA in the nucleus (Shiokawa and Pogo, 1974). The evidence indicates that the genetic defect is in the initiation of protein synthesis; perhaps some of the initiation factors are affected at 36 "C (Hartwell and McLaughlin, 1969). RNA Synthesis in Starved and Nonstarved t,-187 when Protein Synthesis Is Inhibited by Temperature Shift or Cycloheximide. Preliminary experiments indicated that inhibition of R N A synthesis in the t,-187 is observed after 80 min of culturing in a tyrosine-deprived medium. The cells were temperature shifted at this point in order to study the effect of inhibition of protein synthesis on RNA synthesis in amino acid starved cells. As is shown in Figure 2, the changes observed depend upon the labeled R N A precursor used as well as the culture conditions. Thus, if labeled uridine was used, a complex pattern of stimulation and inhibition was observed in both starved and nonstarved cells (Figure 2a). In general this pattern can be divided into three distinct phases: an initial phase of stimulation which is only observed in nonstarved cells; a
-
0
Y
80
I
I
100
120
I
1
140 160 MINUTES
I
I
I
180
200
220
FIGURE 2: Incorporation of [S3H]uridine and [5,6-3H]uracil in the t,-187 cells during temperature shift from 23 to 36 O C . In (a) the cells were resuspended at a density of 3 X lo7 cells/ml in 30 ml of complete (+Ty) and tyrosine-deprived (-Ty) medium for 50 min, at which time 7 pCi of uridine was added. Aliquots (0.5 ml) in duplicate were taken at 80 and 90 min, and 15 ml of each culture was transferred to a 36 O C prewarmed flask. Then 0.5-ml aliquots in duplicate were taken at the intervals indicated. (b) was similar to (a) but the cells' density was 2.5 X lo7 cells/ml, 24 ml was the volume of the culture, and 90 pCi of uracil was added 50 rnin after incubation in a complete (+Ty) and a tyrosine-deprived (-Ty) medium. Arrow, time of temperature shift; 0-0, culture in complete medium at 23 OC; A-A, culture in tyrosine-deprived medium at 23 OC; 0-0,culture in complete medium transferred to 36 OC; m---., culture in tyrosine-deprived medium transferred to 36 OC.
second phase of inhibition which is observed in both starved and nonstarved cells; and a final phase of reinitiation which is also observed in both starved and nonstarved cells. The reinitiation phase of uridine incorporation in nonstarved cells has a lower rate at 36 OC than those maintained at 23 OC. The reverse is true in starved cells (Figure 2a). When labeled uracil was used, inhibition of incorporation by temperature shift was exclusively observed in nonstarved cells (Figure 2b). The degree of inhibition of uracil incorporation was more pronounced than that of uridine in starved cells cultured either at 23 or at 36 OC. Thus, the uridine incorporation of cells cultured at 23 OC and starved for 140 min was approximately half of the incorporation observed in nonstarved cells (Figure 2a). On the contrary, a similar condition produced inhibition in the incorporation of uracil which was about thirty times greater (Figure 2b). A logical conclusion of these observations is that amino acid starvation and inhibition of protein synthesis interfere with RNA metabolism by affecting the RNA synthetic machinery as well as the conversion of uracil, but not of uridine, into UTP and CTP (see below). The changes in the incorporation of labeled uridine when the ts- 187 is temperature shifted resemble the effect of cycloheximide on starved and nonstarved A364A (Gross and Pogo, 1974). This indicates that, when protein synthesis is inhibited either by the addition of this drug or by temperature shift, BIOCHEMISTRY, VOL.
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I121
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bo
100
180 220 MINUTES
Iu)
260
YK)
FIGURE 3: Effect of cycloheximide on the incorporation of [5-3H]uridine in t,-187 cells cultured at 23 OC in complete (a, left) and tyrosine-deprived (b, right) medium. The cells were resuspended at a density of 2 X lo7 cells/ml in 30 ml of complete and tyrosine-deprived medium for 50 min at which time 90 FCi of uridine was added to each culture. Duplicate aliquots (0.5 ml) were taken at 80 and 90 min, and then 12 ml of each culture was transferred to another flask where to each was added cycloheximide (Calbicchem) to make SO $g/107 cells. Aliquots (0.5 ml) in duplicate were taken at the intervals indicated. Arrow, time of cycloheximide addition; 0-0, in the absence of the drug; 0-0, in the presence of the drug.
stimulation in R N A synthesis occurs in starved cells and inhibition of R N A synthesis in nonstarved cells. The experiment described in Figure 3 shows that cycloheximide affects the incorporation of labeled uridine in starved and nonstarved ts-187 in a similar manner to that of the A364A (Gross and Pogo, 1974)-it inhibits this incorporation in nonstarved cells, but stimulates it in starved cells. However, the temperature shift has a peculiar effect which is independent of amino acid starvation. Thus, when the cells are transferred to 36 O C there is an inhibition of uridine ilptake, Le., the inhibitory phase. To further explore this phenomenon, temperature-shift experiments were done in the A364A in which stimulation of protein synthesis is observed immediately after the transfer (Figure 1). Effect of Temperature Shift During the Incorporation of Uridine and Uracil in Starved and Nonsrarued A364A Cells. As with nonstarved ts-187, the transfer to 36 O C produces the three distinct phases-an initial phase of stimulation, a second phase of inhibition, and a final phase of reinitiation in uridine incorporation (Figure 4a). However, there are two important features which differentiate the temperature effect on A364A from t,-187. One is that these three phases are seen in uridine as well as in uracil incorporation (Figure 4b). The other is that the rate of incorporation in the reinitiation phase is greater a t 36 OC than a t 23 OC which results in, as expected, an R N A metabolic rate greater at 36 O C than at 23 "C (Figure 4a, b).i In the experiment described in Figure 4c the effect of temperature shift during uridine incorporation in amino acid starved A364A cells is shown. As with the ts-187, it produces two distinct phases-inhibition and reinitiation of the uridine incorporation (Figure 2a). Again the rate of uridine incorporation in the reinitiation phase is higher a t 36 O C than at 23 O C (Figure 4c). In general the inhibitory phase is much less pronounced when labeled uridine rather than labeled uracil is used. In several experiments this phase is so tenuous that a similar rate of uridine incorporation has been observed a t 23 and 36 OC. In addition, uridine incorporation starts to decline a t about 150
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FIGURE 4: Incorporation of [5-3H]uridine and [5,6-3H]uracil in the A364A cells during temperature shift from 23 to 36 OC and cultured in complete and tyrosine-deprived medium. In (a) the cells were cultured at a density of 2 X lo7 cells/ml in 30 ml of complete medium. Then 140 WCi of uridine was added and 0.5-ml duplicate aliquots were taken at 60 and 70 min. At 70 min, 15 ml of the culture was transferred to a prewarmed 36 OC flask and 0.5-ml duplicate aliquots were taken at the indicated intervals. In (b) conditions were similar to (a) but 170 pCi of uracil was added. In (c) the cells were resuspended in 60 ml of tyrosine-deprived medium at the same density as in (a) and cultured for 30 min. Then 300 j L i of uridine was added to 60 ml of culture and at 70 min 30 ml were transferred to a 36 OC prewarmed flask. 0-0, culture at 23 OC; A---A, culture at 36 OC.
min in cells maintained a t 23 O C (Figure 4a); this is not the case with uracil (Figure 4b). Taking into consideration these observations and the fact that nonlabeled uracil is present when labeled uridine is used, it is evident that there is a preferential utilization of uridine vs. uracil. Thus, the decline in uridine incorporation indicates a diminution of labeled uridine in the medium. The fact that temperature shift either in A364A or ts-187 produces a drastic inhibition in the uptake of uracil rather than uridine strongly indicates that a 36 O C the entry of the base into the UTP and CTP pools is blocked (see below). Effect of Temperature Shift and Starvation on the Four Main Ribonucleotide Pools and the Conversion of Uracil into
CONTROL OF R I B O N U C L E I C A C I D S Y N T H E S I S IN E U K A R Y O T E S
Table I: Effect of Amino Acid Starvation and Temperature Shift on the Nucleoside Triphosphate Po01.~ pmol/ 106 Cells Expt
Strain
Culture Conditions
Temp ("C)
[32P]ATP
1
A364A
2
A364A
Complete medium Minus tyrosine Complete medium Minus tyrosine Complete medium Complete medium Complete medium Complete medium Complete medium Complete medium
23 23 23 23 36b 3@ 23
198 128 188 198 129
A364A A364A ts- 187
ts-187 ts-187 ts-187
[32P]GTP
57 85
36b 36b 36c
15 26
25 24 35 32 13 26 11 9 8 10
255 74 100
[32P]CTP
13 32 10 12 5 9 13
10
[32P]UTP 16 43 8
44 10 7 6 4
7
16
a Fifteen milliliters of A364A cells was grown overnight with 0.4 mCi of H332P04in complete medium to a density of about 2 X lo7 cells/ml. They were then harvested and isolation and determination of the amounts of 32P-labeledribonucleotides were done as explained in Materials and Methods. The values correspond to the average detected in 14 samples of cells cultured in complete medium for 6 rnin (see Figure 5). 10 rnin after transfer to 36 OC. 100 rnin after transfer to 36 OC.
UTP and CTP. As has been indicated previously, temperature shift and starvation appear to affect the conversion of uracil rather than uridine into UTP and C T P pools. The determination of the pool size of the four main ribonucleotides and the rate of conversion of uracil into UTP and CTP as well as uracil incorporation into R N A has been done in order to study this important phenomenon. Temperature shift and tyrosine starvation produce changes in the amount of some of the four main RNA precursors (Table I). In the A364A stringency increases CTP and UTP pools but temperature shift produces no significant changes. In the t,-187 an increase of ATP, CTP, and UTP pools is observed 100 rnin after a temperature shift (Table I). The kinetics of entry of uracil2 into the UTP and CTP pools of the A364A after temperature shift and during stringency are shown in Figure 5. In the control (Figure 5a) there is an initial rapid rise which extends over the first 300 s and is followed by a slower rise, which appears linear for the rest of labeling. The ordinate intercept of the back extrapolate of the linear phase is 3.3 X lo2 dpm/pmol, or approximately 1.7% of the input specific activity of the uracil. Both the kinetics of entry and the low rate of conversion indicate that in yeast there is a very large cellular UTP pool. Entry into the C T P pool is much slower and does not exhibit the initial exponential phase (Figure 5a). The kinetics of entry in A364A cells after 10 rnin of transfer to 36 OC and 50 rnin of tyrosine starvation are shown in Figure 5b,c, respectively. In both, the initial rise is less rapid than in control cells and the specific activity is reduced to approximately half that of the control (1.2 X lo2 dpm/pmol). An extensive reduction in the conversion of labeled uracil into the C T P pool is observed. A dramatic reduction in the entry of uracil into UTP and C T P is seen when protein synthesis and R N A synthesis are blocked, i.e., t,-187 cultured at 36 OC (Figure 5d). Since stringency and temperature shift produce an increase in the UTP and CTP pools, the logical explanation of the inhibition of the conversion of uracil into UTP and CTP is by a feedback mechanism. The experiments done with the incorporation of labeled uridine into total R N A indicate that temperature shift, strin~~
* This is a pyrimidine auxotroph in which the genetic defect lies in the enzyme orotate reductase (EC 1.3.1.14) which converts dihydroorotate into orotate (de Robichon-Szulmajster and Surdin-Kerjan, 1971).
-
5.0r
5
4.0-
.-
U
b UTP
Time (seconds) FIGURE 5: Rate of conversion of [5,6-3H]uracil into total pyrimidine triphosphate pools in A364A cells cultured in complete medium at 23 O C (a), tyrosine-deprived medium at 23 O C (b), 10 min after transfer to 36 OC (c), and t,-187 cells 100 min after transfer to 36 O C (d). Uracil (300 MCi, specific activity 8.9 mCi/pmol) was added to 15 ml of 32P-labeled cells cultured at a density of 2 X lo7 cells/ml. Samples were withdrawn at the indicated intervals, and specific activities of the intracellular UTP and CTP pools were determined as described (Materials and Methods). The dashed line represents the extrapolate of the linear phase to the intercept of the ordinate. This determines the specific activity of the UTP pool.
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Table - TI: Effect of Inhibition of Protein Synthesis by Temperature Shift on RNA Synthesis of ts-187 Isolated Nuclei." Assav Conditions (Dm01 of UMP/IO* Nuclei) a-Amanitin-Resistant
Expt 1
2
Incubation Time (min) 5
30 60 5 30
23 OC
36 OC
36 OCJ23 OC
130 143 187
40 33 22 16 39
0.30
0.23 0.12 0.62 0.22
32
0.10
123
176 311
60
a-Amanitin-Sensitive 36 OCJ23 OC
23 "C
36 O
34
40
103 32 75 104
57
I .80
59 52
0.80
77
33 18
C
___.____
1.2
0.43 0.17 0.50
ts-187 spheroplast suspension (400 ml) at a density of 5-6 X lo7 cells/ml was incubated at 23 OC for 1 h. Then 200-ml aliquots of fresh medium prewarmed to 23' and 50 "C were added to equal volumes of spheroplast cultures and at the indicated intervals the spheroplastswere harvested, nuclei isolated, and RNA syntheses assayed in triplicate as described in Materials and Methods. The stepwise addition of a prewarmed medium (50 " C ) to a 23 O C culture increased the temperature to 36 "C in less than 1 min. a-Amanitin-sensitive activity was calculated by the difference between the amounts of [5-3H]UMPincorporated in the absence and presence of 30 kg of the toxin. The a-amanitin-resistant activity is the amount of [5-3H]UMPincorporated in the presence of the toxin.
-
rated than the cellular UTP pool. This is indicated by the fact that the line in the experiments described in Figure 6 does not 7.0extrapolate through the origin, and linearity in the rate of incorporation of U M P into R N A is reached in approximately 6.0u one-third the interval necessary to saturate the cellular U T P rn pool (Figure 5 ) . From the data of the experiments described in Figures 5 and 6 , it is estimated that the amount of [3H]UMP incorporated into RNA at the time of saturation of the nuclear pool (120 s) is 1.5% of the amount of [3H]UTPin the cellular pool. These observations are consistent with the concept that there is 8 small and preferentially labeled nuclear UTP pool in yeast. Two distinct ribonucleotide pools, one in the nucleus and another in the cytoplasm, have been described in other I 1 eukaryotes (Plagemann, 1972). 63 120 180 240 300 Effect of Temperature Shift on R N A Synthesis in Nuclei T I M E (seconds) Isolated from Different Strains of Yeast. As has been demFlGlJRE 6: Rate of incorporation of UMP into total RNA of A364A cells onstrated before, amino acid and glucose starvation and inhicultured at 23 OC in complete medium. The H332P04labeling was performed as described in Figure 5 but the final cell density was 4 X IO7 bition of protein synthesis by cycloheximide produce a dracells/ml. The [5,6-3H]uracillabeling was performed by adding 1.6 mCi matic reduction in the synthesis of R N A in isolated yeast nuclei (specific activity 100 mCi/jmol) into 40 ml of culture. The raw data have (Gross and Pogo, 1974). It appears that nuclear R N A polybeen corrected for the distribution of label between UMP and CMP to merase activities, in contrast to the same activities detected in obtain radioactivity in UMP alone (Figure 5 ) . These values were also corrected for the specific activity of the UTP pool ( [5-3H]UTP/[32P]UTP) crude extracts and after chromatographic fractionation, are to obtain pmol (of UMP/108 cells incorporated into RNA). highly sensitive to nutrient deprivation as well as to antibiotics which suppress the protein synthetic machinery. In accord with these observations it has been found that culture conditions gency, and suppression of protein synthesis do not significantly prevent the conversion of uridine into UTP.3 Therefore, in these. have marked effects on both the a-amanitin-resistant R N A polymerase activity, which is assumed to be involved in rRNA yeast strains the phosphorylation of uridine, which can take synthesis, and the a-amanitin-sensitive R N A polymerase acplace through the "salvage mechanism", does not appear to tivity, which presumably synthesizes DNA-like RNA. Thus, be under feedback inhibition by UTP. both activities increase in a similar manner during the first 60 Although these observations are concerned with the total min of recovery after the Glusulase treatment. (During this cellular pool of nucleotides, it is assumed that similar changes treatment the spheroplasts are in starved conditions.) Thereshould occur in the nuclear pool. It appears that the nuclear after there is a preferential increase of a-amanitin-resistant pool of UTP is preferentially labeled and more rapidly satuactivity (not shown). This activity is much higher in nuclei isolated from cells in early or middle logarithmic growth than from cells in stationary or lag phase of growth (unpublished Kinetics of uridine conversion into UTP and CTP showed a similar results). pattern to that of uracil but the increase was not as rapid. The specific activity of the total UTP pool was approximately one order of magnitude The experiments described in Table I1 were done with t,-187 less than that obtained with uracil. This low specific activity can be due nuclei harvested a t different stages of growth. In addition, to the exclusive entry of uridine into the small yeast nuclear UTP pool. The parallel experiments of [5-3H]uridine incorporation were done preferential utilization and the greater incorporation of the nucleoside vs. with spheroplasts maintained a t 23 'C and shifted to 36 O C . the base strongly support this assumption (Figure 4). A similar observation has been reported in Novikoff hepatoma cells (Plagemann, 1972). Similar results to those observed in cells were always obtained, 8.0
,
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~
Table 111: Effect of TemDerature Shift on R N A Synthesis in Isolated A364A and S288C Nuclei." Assay Conditions (pmol of UMP/108 Nuclei)
I Strain
Expt
A364A
1
A364A
2
A364A S288Cd
3
4
Incubation Time (min) 5 30 60 5 30 60 10 20
a-Amanitin-Resistant
l
a-Amanitin-Sensitive
23 OC
36 OC
36 '(2123 OC
23 OC
36 OC
36 OC/23 OC
140 150 170 120 150 200 128 104
26 62 126 90 120 170 64b 66
0.18 0.41 0.74 0.75
90 74 125 80 74 100 175 84
34 31 84 70 76 120 106b 60
0.38 0.42 0.67 0.88 1.02 1.20 O.6Oc 0.7 I
0.80 0.85 O.5Oc 0.63
Similar to Table I1 but in A364A and S288C spheroplast cultures. In experiment 5 the temperature shift was to 33 OC. Cultured at 33 OC. Ratio 33 OC/23 OC. Due to contamination by total cells the amount of DNA per los nuclei represents the amount obtained in the supernatant of a sodium dodecyl sulfate treated aliquot (1% v/v) and centrifuged at 10 OOOg for 30 min. (I
i.e., permanent inhibition in the incorporation of labeled uridine at 36 OC (not shown). Immediately after the ts- 187 spheroplasts are transferred to 36 OC there is inhibition of a-amanitin-resistant activity (Table 11). Later on and to a lesser extent the a-amanitinsensitive activity is inhibited, Finally, 1 h after having been transferred to 36 OC,there is a pronounced inhibition in both nuclear R N A polymerase activities. These findings indicate that inhibition of protein synthesis has a profound effect on the R N A synthetic machinery and that the inhibition of RNA synthesis in isolated nuclei from cycloheximide-treated cultures or injected animals is not due to a pecuiiar effect of the drug but because it inhibits protein synthesis (Gross and Pogo, 1974; Muramatsu et al., 1970; Yu and Feigelson, 1971).4 Evidently R N A synthesis in isolated nuclei is highly sensitive to any inhibition of protein synthesis induced by starvation, cycloheximide or temperature shift in the t,-187. Data from similar experiments in nuclei isolated from A364A and S288C spheroplasts are presented in Table 111. In experiment 5 the temperature shift was made to 33 O C . The fact that temperature shift produces a transitory but significant inhibition of RNA synthesis in isolated nuclei in these yeast strains indicates that the early inhibition observed in nuclei isolated from t,-187 can be a phenomenon attributable to the effect of increasing the temperature per se. That the &-187,but not the A364A, does not recover after 1 h at 36 OC indicates that this inhibition or late inhibition in the nuclear R N A polymerase is due to the inhibition of protein synthesis. The experiment also indicates that a-amanitin-resistant activity is more affected than a-amanitin-sensitive activity. As with the experiment of [5-3H]uridine incorporation, the increase in temperature produces two different effects at the level of the nucleus. The early inhibition is independent of the strain of yeast used and is not related to inhibition of protein synthesis. The late inhibition of nuclear R N A polymerase activities, however, is directly related with the inhibition of protein synthesis since it only occurs in the t,-187. Effect of Temperature Shift on the Activities of Isolated R N A Polymerase. As has been indicated before, R N A polymerase activities detected in crude extract and with exogenous ~
~
~~~~~
~
The degree of inhibition in uridine incorporation and nuclear RNA polymerase activities is not directly comparable since in the former there is a reinitiation of RNA chains and in the latter only elongation and termination occur.
DNA template are not affected either by prolonged periods of starvation or inhibition of protein synthesis by cycloheximide. Furthermore, no interconversion of the different species of enzymes has been detected in any of these culture conditions (Gross and Pogo, 1974). Although recent studies have shown that a duplex structure containing unpaired gaps is an excellent template for yeast R N A polymerases, mainly I (or A) and I1 (or B) (DeA16e et al., 1974), the status of the natural template of these enzymes is unknown. Therefore, quantitation of the RNA polymerases isolated from yeast, like those isolated from any other organism by the determination of their activities with exogenous template, has this limitation. In addition one should consider that the assay is done in a crude extract. Nevertheless the study of the activities of isolated enzymes and their fractionation in DEAE-Sephadex chromatography can be a useful procedure to detect gross changes in amounts and/or biochemical properties provided that an identical assay is used for enzymes isolated from the same cells cultured under different and well-defined conditions. This is, so far, the only available procedure for the quantitation of RNA polymerases in cellular extracts. Table IV shows the RNA polymerase activities detected in crude extracts of the t,-187 spheroplasts cultured at 23 and 36 OC for 1 h. Complete shut-off of protein synthesis has no effect on the a-amanitin-resistant and -sensitive enzymatic activities assayed with exogenous template. This is similar to the results obtained in starved or cycloheximide-treated A364A spheroplasts (Gross and Pogo, 1974). In experiments 6 and 7 the enzymes were extracted with a buffer that contains 0.5%each of Triton X-100 and saponin, detergents regularly used to isolate yeast nuclei. Since no significant difference is observed in the presence and absence of these detergents, it is evident that they do not affect the R N A polymerase activities. In addition, if the detergents are added to the enzymes fractionated and purified by DEAE-Sephadex chromatography, no interference with the RNA synthetic process is observed (not shown). Fractionation of these crude extracts by DEAE-Sephadex chromatography is shown in Figure 7. As in the A364A strain, elution of the column by an ammonium sulfate gradient resolves five peaks which are not altered by prolonged periods of inhibition of protein synthesis (Gross and Pogo, 1974). To analyze the total amount and relative proportion of each enzyme species, eluates of the individual peaks were pooled and assayed with the appropriate DNA template at saturation BIOCHEMISTRY, VOL.
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Table IV: R N A Polymerase Activities of t,-187 Crude Extract.O pmol of U M P / IO8 Cellsb
pmol of UMP/pg of Proteinb Expt
Incubation Time (min)
Culture Conditions
1
30
23 OC
2
60
23 OC
3c
60
23 OC
4
30
36 OC
5
60
36 OC
a-Amanitin-Resistant
a-Amanitin-Sensitive
a-Amanitin-Resistant
a-Amanitin-Sensitive
0.67 (0.65-0.70) 0.63 (0.60-0.70) 0.75 (0.70-0.80) 0.57 (0.55-0.60) 0.62 (0.50-0.75)
0.55 (0.40-0.70) 0.75 (0.60-0.90) 0.80
246 (212-280) 21 1 (200-23 1) 310 (296-324) 226 (201-252) 251 (2 10-300)
212 (138-287) 260 (220-302) 318 (308-329) 238 (154-322) 266 (241 -295)
0.57 (0.40-0.75) 0.72 (0.65-0.75)
t,-187 spheroplasts were incubated for 1 h at a density of 4 X lo7 cells/ml and divided into 500-ml aliquots. Then 50-ml aliquots of fresh medium prewarmed to 23O and 50 OC were added to each aliquot and cultured further as indicated. The enzymes were isolated as explained in Materials a n d Methods and duplicate aliquots of 10-20 pg of protein from the high-speed supernatant were assayed in the presence of 0.25 mM [ S 3 H ] U T P (specific activity 20 cpm/pmol) and 0.1 M ammonium sulfate which is the optimal concentration for the amount of crude extract used with all the other components of the assay mixture. For the a-amanitin-resistant reaction, 2 pg of a-amanitin was added, which gives maximum inhibition. Denatured D N A was used in all the assays and the difference between the amounts of [5-3H]UMP incorporated in the absence and presence of the toxin gives the amount corresponding to the a-amanitin-sensitive activity. a-Amanitin-resistant activity is the amount of [5-3H]UMP incorporated in the presence of the toxin. Picomoles of U M P / lo8 cells were calculated as explained in Materials and Methods. Average of two or three experiments; minimum and maximum values in parentheses. Extracted in a buffer containing 0.5% each saponin and Triton X-100.
"1
9p
a
-06
Table V: R N A Polymerase Activities of t,-187 Fractionated by DEAE-Sephadex Chromatography.'
IC
25
05
2 ot
04
I 51
Culture Conditions : 23 "C ;06
IC
$c
5
I'
10-52 z
20 2.01
404g
I
I5t I .o/ I
0.5-
OI
, ,
) '
,v, 1" , ; , ' $dl
20
40
11 60
\
80
ml
FIGURE 7:
Fractionation of t,-187 RNA polymerases by DEAE-Sephadex chromatography. After the spheroplasts were recovered from Glusulase treatment, two aliquots were centrifuged and resuspended at a density of 2.5 X IO' cells/ml in 1.5 I. of complete medium. Then the spheroplast cultures were incubated for another hour, one at 23 O C (a) and the other at 36 OC (b). The enzymes were extracted, fractionated, and assayed (see Materials and Methods) with 0.05 mM [5-3H]UTP (specific activity 150 cpm/pmol).
substrate concentrations (Table V), As in the A364A, no significant difference is observed in enzymes obtained from spheroplasts cultured in the absence of protein synthesis. The combined polymerase complex I (or A) and I11 (or C ) represent the a-amanitin-resistant species, while polymerase I1 (or B) represents the a-amanitin-sensitive species detected in the crude extract (Tables IV and V). It is seen that after fractionation the enzymes obtained from spheroplasts cultured at 36 O C have a higher activity than those extracted from spheroplasts cultured at 23 OC (Table V). This is more apparent in polymerase 11 (or B) than in the other species. The significance of this observation is difficult to assess since recovery from the column is in the range of 80-8596 of the total input. In any case, their elution patterns are similar, indicating
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36 OC
pmol of UMP/108 Cellsh Polymerase I
Polymerase I1
Polymerase I11
79 (75-83) 103 (86- 120)
170 (170-171) 247 (207-295)
91 (80- 103) 109 (67-1 33)
a Each chromatographic enzyme fraction (see Figure 7 ) was p l e d , and 50-111 aliquots were assayed with 20 gg of DNA in the presence of all the components of the assay mixture as explained (Gross and Pogo, 1974). The polymerase I complex was assayed with native DNA, with no additional ammonium sulfate and 0.125 mM [ 5 3H]UTP (specific activity 50 cpm/pmol). Polymerase I1 was assayed with denatured DNA, 0.08 M ammonium sulfate, and 0.25 mM [ S 3 H ] U T P (specific activity 25 cpm/pmol). Polymerase I11 was assayed with denatured DNA, 0.08 M ammonium sulfate, and 0.25 m M [5-3H]UTP (specific activity 25 cpm/pmol). The values represent the total amount recovered from the column. Average of two or three experiments; minimum and maximum values in parentheses.
that inhibition of protein synthesis does not produce major distortions of these enzymes. Any study of the isolated nuclear RNA polymerases in yeast has to take into consideration the purity of the yeast nuclear fraction. Although the presence of some intact cells does not represent a serious inconvenience when nuclear RNA polymerase activities are determined in intact nuclei (the cells do not participate in these reactions), they can be an important source of contamination when these enzymes are extracted from the nuclei. Therefore, our studies of the RNA polymerases extracted from nuclei were done only with A364A nuclear preparations which did not contain cells. Since glucose starvation produces similar effects on the nuclear R N A polymerases to those of inhibition of protein synthesis either by temperature shift in the t,-187 or the addition of cycloheximide, the nuclear fractions were obtained from A364A spheroplasts cultured in the presence and absence
CONTROL OF RIBONUCLEIC ACID SYNTHESIS IN EUKARYOTES 25 20.
~~
Table VI: R N A Polymerase Activities of A364A Crude Nuclear Extract Obtained from Glucose-Starved and Nonstarved SDheroalasts'rb Culture Conditions
6.3
49
58
2.5
4.2
24
36
0.2
Table VII: R N A Polymerase Activities of A364A Nuclear Extract Fractionated in DEAE-Sephadex Chromatography.(1 pmol/108 Nuclei Polymerase I Polymerase I1 Polymerase 111
18 9
33 14
7.0 7.5
Each chromatographic enzyme fraction (see Figure 8) was pooled and assayed as explained in Table V.
of glucose (Gross and Pogo, 1974). The results indicate that glucose starvation produces a significant diminution in both a-amanitin-resistant and a-amanitin-sensitive activities (Table VI). This correlates with a similar inhibition observed when the enzymes are assayed in intact nuclei with their physiological templates (Gross and Pogo, 1974). However, the degree of inhibition observed with the endogenous template is greater than when the enzymes were isolated and assayed with exogenous template, indicating that in the nucleus the RNA polymerases may exist bound and unbound to the DNA template. This observation concurs with the recent detection of two discrete functional pools of nuclear RNA polymerase in rat liver and lymphosarcoma (Lampert and Feigelson, 1974). In Figure 8 a typical fractionation of the A364A R N A polymerases extracted from nuclei of glucose-starved and nonstarved spheroplasts is shown. Except for a diminution in practically all the enzyme species (Table VII), the patterns are similar in both culture conditions. In the yeast nuclei about 30 and 15% of the total cellular enzymes are recovered in nonstarved and starved conditions, respectively. This low recovery can be attributed to leakage due to the use of detergents in the purification of the nuclear fraction, or may reflect a true nuclear-cytoplasmic enzyme distribution. It remains to be resolved which of these alternatives is real. It is important to mention that, although our results indicate two discrete pools of nuclear enzymes, prolonged
W
2 I
0.1
C 0.5
0
5
3
x
0)
20
0.4
5
I .o 0.I
20
(1
Plus glucose Minus glucose
-
10.4
0.5
Spheroplasts were incubated at a density of 5 X lo7 cells/ml, then divided into 500-ml aliquots and cultured for 60 min. One aliquot was centrifuged and the spheroplasts were resuspended in 1.2 I. of glucose-deprived medium at a density of 2 X lo7 cells/ml. The other aliquot was diluted with complete medium to the same density. After 1 h of incubation under these conditions the spheroplasts were harvested, the nuclei isolated and the nuclear R N A polymerases extracted as explained (Materials and Methods). The high-speed supernatant was assayed in the presence of 0.25 m M [ S 3 H ] U T P (specific activity 20 cpm/pmol) and 0.1 M ammonium sulfate, which is the optimal concentration for the amount of crude extract used, with all the other components of the assay mixture. For amounts of a-amanitin, DNA, and calculation of a-amanitin-resistant and -sensitive activities see Table IV. Average of 2 experiments.
Culture Conditions
I
0.3 2
a-Amanitin- a-Amanitin- a-Amanitin- a-AmanitinResistant Sensitive Resistant Sensitive 4.8
I
n l
pmol of UMP/pg of proteinb pmol of UMP/108 Cellsb
Plus glucose Minus glucose
0.5 a
40
60
80ml
8: DEAE-Sephadex chromatographic fractionation of RNA polymerases extracted from A364A nuclei. After the spheroplasts were recovered from Glusulase treatment, two aliquots were centrifuged and resuspended at a density of 3 X IO7 cells/ml in 2 1. of complete (a) and glucose-deprived medium (b). Then the cultures were incubated for another hour. The nuclei were prepared and the nuclear enzymes were extracted, fractionated, and assayed (Materials and Methods) with 0.05 mM [S3H]UTP (specific activity 100 cpm/pmol). Notice that in this chromatographic fractionation the resolution of enzyme subspecies is much better than when enzymes are isolated from total spheroplasts (Figure 7). Polymerase I includes all the fractions eluted up to 0.2 M ammonium sulfate, polymerase I1 includes all the fractions from 0.2 M to 0.28 M ammonium sulfate, and polymerase 111includes all those above 0.28 M, as indicated by the brackets. Assay of polymerase I fractions in the presence of 2 fig of a-amanitin showed 10% inhibition of these enzyme species, polymerase I1 fractions 90%. and none for polymerase I11 fractions. FIGURE
periods of nuclear incubation at 4 "C in a buffer identical with the one used for the RNA synthesis in isolated nuclei do not produce a significant leakage of the DNA-unbound enzyme (unpublished results). It may be that the DNA-unbound enzymatic fraction is firmly attached to some nuclear elements. Discussion The experiments reported here indicate that inhibition of protein synthesis affects yeast RNA metabolism by a direct effect on the RNA synthetic machinery. Although inhibition of protein synthesis affects the processing and accumulation of rRNA species in eukaryotes (Burdon, 197 l), it is also evident that the transcriptional apparatus is highly sensitive to the protein synthetic machinery. The nuclear RNA polymerase activities, Le., RNA synthesis in isolated nuclei, are the main targets when protein synthesis is inhibited. Since a similar effect has been observed when cycloheximide is added to the culture medium (Gross and Pogo, 1974), it is concluded that this inhibitory effect of the drug is due primarily to its inhibition of protein synthesis and not to some unknown effect on the nuclear enzymes. With the use of the t,- 187, this important point has been resolved. The coupling of RNA and protein synthesis is nonstarved t,-187 cells cultured at 36 OC is a further corroboration that stringency exists in nucleated cells. The fact that uncoupling is produced by the addition of cycloheximide to amino acid starved t,-187 cells cultured at 23 O C or by the transfer of these cells to 36 OC is further evidence that relaxation occurs in nucleated cells. This confirms our previous work with the A364A strain as well as similar results in glucose shift down experiments done in other yeast strains (Gross and Pogo, 1974; Foury and Goffeau, 1973). It appears that entrance of uridine into the UTP and CTP pools is not feedback inhibited by these ribonucleotides in yeast. This conversion takes place through an enzymatic reaction which involves uridine-cytidine kinase and ATP. Very little BIOCHEMISTRY, VOL.
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is known about the regulation of this enzyme in other eukaryotes except that the one isolated and purified from rat hepatoma appears to be subject to allosteric inhibition by CTP and UTP (Orengo, 1969). If this is the case for other animal cells, then this may explain why relaxation has never been clearly observed in HeLa cells (Smulson and Thomas, 1969). In the yeast strains used for our experiments it appears that the main route of uracil entrance into the ribonucleotide pool is by way of a single step phosphoribosyltransferase reaction specific for the base (de Robichon-Szulmajster and Surdin-Kerjan, 1971). Although little is known about the regulation of the yeast uracil phosphoribosyltransferase (EC 2.4.2.9) a similar enzyme, the orotate phosphoribosyltransferase (EC 2.4.2. lo), which is specific for orotate, is feedback inhibited by UTP in yeast (de Robichon-Szulmajster and Surdin-Kerjan, 197 1). This explains the inhibition in the conversion of uracil into UTP and CTP that starvation and inhibition of protein synthesis produce (Figures 5b,c). It also explains why relaxation cannot be observed when uracil is used as labeled R N A precursor (Figure 2 and Roth and Dampier, 1972). The temperature-shift experiments indicate that the nuclear RNA polymerases can be inhibited by other means in addition to the inhibition of protein synthesis. Thus, immediately after the temperature shift in A364A, S288C, or t,-187 there is inhibition (early inhibition) of the nuclear enzymes. Since this effect is seen in the presence of active protein synthesis (A364A and S288C), it supports the assumption that temperature shift per se can exert a control on the RNA synthetic machinery. This early inhibition is also observed when the temperature is increased to 33 “C. There is no doubt that the late inhibition in the nuclear RNA polymerase activities, only observed in the t,-l87, is due to the inhibition of protein synthesis. Thus, it does not occur in the A364A, and after 1 h at 36 OC there is a higher level of activity than at 5, 10, or 30 min after the temperature shift. Furthermore, similar values are observed between spheroplasts maintained at 23 OC and those shifted and kept for 1 h at 36 OC (Table 111). The release from this inhibitory phase can be compared to a relaxed phenomenon but of the nuclei or “nuclear relaxation”. Since in order to have this “nuclear relaxation” it is necessary to have both active protein synthesis and nonstarving conditions, it represents the opposite of cellular relaxation. Thus, cellular relaxation, that is to say uncoupling of RNA and protein synthesis in the cell, is a phenomenon observed in the absence of protein synthesis (Figures 2 and 3). This paradoxical effect between the inhibition of nuclear RNA synthesis and stimulation of R N A synthesis in the cells leads us to postulate that modulation of RNA synthesis in yeast and perhaps in other eukaryotes takes place by the interplay of at least two factors-one has the ability to switch on and the other to switch off the RNA synthetic machinery (Gross and Pogo, 1976). The DNA-dependent RNA polymerases are not the ratelimiting factors in RNA synthesis, and this is clearly shown when these enzymes are isolated from the t,-l87 spheroplasts cultured at 36 OC. Although in this condition there is no protein synthesis, no significant changes are observed in the activities of these enzymes. This is consistent with similar results obtained with the RNA polymerases extracted from the A364A strain cultured in the absence of tyrosine or glucose as well as in the presence of cycloheximide (Gross and Pogo, 1974). Similar results have been reported for hepatic R N A polymerases (Benecke et al., 1973). A recent observation in a mutant rat myoblast cell of an a-amanitin-resistant RNA polymerase I1 (or B) indicates that
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this enzyme may be self-regulated. Although no indication in de novo enzyme synthesis has been reported, it is observed that it takes 56 h after removal of the inducer of this enzyme (aamanitin) to return the a-amanitin-resistant RNA polymerase I1 (or B) to the prechallenge level (Somers et al., 1975). It is concluded, therefore, that the observation of low turnover of RNA polymerases in yeast can be extended to other nucleated cells. It is evident that nuclear R N A polymerases are highly sensitive to environmental changes. This is not only true in yeast and ascites but also in rat liver and lymphosarcoma (Muramatsu et a]., 1970; Sajdel and Jacob, 1971; FranzeFernQndez and Pogo, 197 1; Franze-Fernbndez and Fontanive-Sengiiesa, 1973; Gross and Pogo, 1974; Yu and Feigelson, 1971; Lampert and Feigelson, 1974). In addition, the inhibition of these enzymes has been observed when they were extracted from the yeast nucleus and assayed with an exogenous and nonphysiological template. Notwithstanding, this condition exhibits much less inhibition than when the RNA polymerases are assayed in situ. Furthermore, no significant changes in their chromatographic patterns and proportions have been observed between RNA polymerases isolated from yeast nuclei obtained from starved and nonstarved cells. The results indicate that the reduction in the nuclear RNA polymerase activities is probably due to a net diminution of the number of enzyme molecules bound to the natural template. In other words, 3. diminution in the R N A synthesis in isolated nuclei indicates a restriction in the nuclear DNA template. This is consistent with the notion that RNA chain elongation and termination are the only reactions that takes place in isolated nuclei (Pogo et al., 1966; Pogo, 1969). It remains to be seen if in yeast there are nuclear and cytoplasmic pools of RNA polymerases. Two discrete functional nuclear pools, one bound and the other unbound to the template, have been recently reported for nuclear RNA polymerase in rat liver and lymphosarcoma (Lampert and Feigelson, 1974). As in yeast, the DNA-bound pool is affected by the addition of cycloheximide. Since the procedure used to purify yeast nuclei could easily produce nuclear leakage of the DNA-unbound enzymes, it is not possible to discern between a real or an artificial depletion of the DNA-unbound RNA polymerases when protein synthesis is inhibited or when the cells are cultured in starving conditions. Acknowledgments We wish to thank Ms. Valerie Zbrzezna for her expert technical assistance. References Benecke, B. J., Ferencz, A., and Seifart, K. H. (1 973), FEBS Lett. 31, 53-58. Brachet, J. (1957), in Biochemical Cytology, Academic Press, New York, N.Y., p 226. Burdon, R. H. (1971), Prog. Nucleic Acid Res. Mol. Bioi. 11, 33-79. Burton, K. (1953), Biochem. J . 62, 315-323. Chen, J . H., Weissman, S. M., and Lengyel, P. (1972), Biochem. Biophys. Res. Commun. 46, 785-789. Crampton, C. F., Lipshitz, R., and Chargaff, E. (1954), J . Bioi. Chem. 206, 499-510. de Robichon-Szulmajster, H., and Surdin-Kerjan, Y. ( 1 97 l ) , Yeasts 2, 335-418. DezeICe, S., Sentenac, A., and Fromageot, P. (1974), J . Bioi. Chem. 249. 5971-5977.
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Natl. Acad. Sci. U.S.A. 56, 550-557. Pogo, A. O., Littau, V. C., Allfrey, V. G., and Mirsky, A. E. (1967), Proc. Natl. Acad. Sci. U.S.A. 57, 743-750. Pogo, B. G.-T. (1972), J. Cell Biol. 53, 635-641. Roth, R. M., and Dampier, C. (1972), J. Bacteriol. 109, 773-779. Rubin, A. D., and Cooper, H. L. (1969, Proc. Natl. Acad. Sci. U.S.A. 54, 469-476. Ryan, A., and Borek, E. (1 97 l), Prog. Nucleic Acid Res. Mol. Biol. 1 1 , 193-228. Sajdel, E. M., and Jacob, S. T. (1971), Biochem. Biophys. Res. Commun. 45, 707-7 15. Somers, D. G., Pearson, M. L., and Ingles, C. J. (1979, Nature (London),253, 312-374. Smulson, M. E., and Thomas, J. (1969), J. Biol. Chem. 244, 5 309-53 12. Stent, G. S., and Bremer, H. (1961), Proc. Natl. Acad. Sci. U.S.A. 47, 2005-2014. Tata, J . R. (1966), Nature (London),212, 1312-1314. Travers, A., Kamen, R., and Cashel, M. (1970), Cold Spring Harbor Symp. Quant. Biol. 35, 4 15-418. Tsukada, K., and Lieberman, I. (1965), J. Biol. Chem. 240, 1731-1736. Winslow, R. M., and Lazzarini, R. A. (1 969), J. Biol. Chem. 244, 1128-1138. Y u , F. L., and Feigelson, P. (1971), Proc. Natl. Acad. Sci. U.S.A. 68, 2177-2180.
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