ARCHIVES
OF BlOCHEMIS’IRY
Control
AND
BIOPHYSICS
of the Formation
Ribonucleic
of
(1975)
of Ribonucleic
Acid in Yeast: Synthesis
Acid in a Nuclear Fraction carlsbergensis
SIWG Department
167, 322-334
R.
Biological
DE
KLOET
Science,
WILLIAM
AND
Florida Received
State May
University,
of
of Saccharomyces
R. BELTZ Tallahassee,
Florida
32306
23, 1974
A study has been made of the nature of the RNA synthesized by isolated yeast nuclei. At 20°C the product consists of high molecular weight RNA sedimenting between 10 and 3OS, including polyadenylic acid containing messenger RNA. At 30°C the synthesized material is mainly of low molecular weight. Optimal conditions for synthesis include the presence of magnesium ions (0.01 M). Manganese ions were found to inhibit very strongly. Nuclei of yeast protoplasts incubated in the presence of amino acids were found to be up to ten times more active than nuclei isolated from starved protoplasts. Yet the activity of the isolated enzymes remained unchanged demonstrating that the activity instead of the actual amount of the enzymes was altered.
The mechanism by which the formation of ribonucleic acid, in particular ribosomal ribonucleic acid is regulated has been extensively investigated in procaryotes, especially Escherichia coli. Genetic analysis has shown that the formation of ribosomal RNA in this organism is under the influence of the RC gene, which in the wild type state correlates the rate of synthesis of ribosomal RNA with the availability of a required amino acid (1, 2). Recent investigations have shown that the RC gene affects the translational apparatus in E. coli (3, 4) and controls the level of a nucleotide 3’-diphosphoguanosine-5’diphosphate (ppGpp)’ (4, 5). The actual involvement of this nucleotide in the biosynthesis of RNA is however still under investigation. No such investigation has been possible in eucaryotes, no RC gene has been identified and the presence of ppGpp has not been reported (6). Yet eucaryotes like yeast show a definite correlation between the ’ Abbreviations used: 3’-diphosphoguanosine-5’diphosphate, ppGpp; 0.02 Tris-HCl buffer pH 7.4, 0.002 M MgCl,, 0.002 M mercaptoethanol, 0.0002 M EDTA, TGMEM; and dimethylsulfoxide, DMSO. 322 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
rate of RNA synthesis and the nature of the growth medium (7, 8, 9), and the addition of amino acids to a minimal medium or the addition of a required amino acid results in a dramatic increase in the rate at which ribosomal RNA is formed. A fundamental difference between procaryotes and eucaryotes is found in the nature of the enzymes which transcribe DNA into RNA. In E. coli a single DNA dependent RNA polymerase occurs which is responsible for the formation of messenger as well as ribosomal RNA (10, 11, 12). In eucaryotes the formation of RNA is carried out by several enzymes which show a distinct nuclear localization and which differ in a number of biochemical aspects. Since one of these enzymes is found in the nucleolus, this enzyme is most likely responsible for the formation of ribosomal RNA, whereas the one or more enzymes found in the nucleoplasm are probably responsible for the transcription of messenger sequences (13-18). Although it has not been possible to fractionate yeast nuclei into similar nucleolar and nucleoplasmic fractions, the similarity in elution patterns of the enzymes upon DEAE Sephadex
RNA
SYNTHESIS
chromatography as compared to the profile obtained from other eucaryotes with more easily identifiable nuclear subfractions (19, 20, 21) suggests strongly that yeast ribosomal and messenger RNA formation is also carried out by different enzymes, very similar to those in higher eucaryotes. The presence of different enzymes for the formation of ribosomal and other RNA species makes an independent control mechanism for the formation of ribosomal RNA very feasible. On the other hand many data (22, 23) suggest that a major regulatory step in the formation of at least ribosomal RNA occurs through the regulation of the maturation process. Inhibition of the formation of ribosomal RNA in yeast by a number of different agents like cycloheximide (8, 24), 5-fluorouracil (25), and heat (26) all give rise to the accumulation of precursor stages of ribosomal RNA. A major prerequisite for the understanding of the mechanism by which the formation of ribosomal RNA is regulated in procaryotes and eucaryotes is the preparation of a cell free system in which this process can be adequately studied. It is the intention of this paper to describe some of the characteristics of a cell free system derived from yeast protoplasts which still reflects the RNA synthesizing capacity of the whole protoplasts from which it has been derived. Several papers have been published dealing with this subject (27, 28, 29). In this paper we will deal especially with the nature of the RNA synthesized in this system as well as with some of the aspects of the mechanism by which the formation of RNA is controlled in yeast. MATERIALS
AND
METHODS
Strains, Saccharomyces carlsbergensis (N.C.T.C. 74) was used for this investigation. Growth of the organism as well as the conversion of logarithmic cells into protoplasm with snail enzyme has been described before (30). Chemicals. [5--*H]Uridine triphosphate (sp act 15 Wmmole), [8-3H]guanosine triphosphate (sp act 20 Ci/mmole) were purchased from Schwarz-Mann, Orangeburg, NJ. y-32P-labeled adenosine triphosphate, (sp act 68 Ci/mmole) r-32P-labeled guanosine triphosphate (sp act 52 Ci/mmole) and gSH-labeled adenine (sp act 20 Ci/mmole) were products of I.C.N.
IN YEAST
NUCLEI
323
All other chemicals were the regular commercially available brands, highest grade of purity. Incubation of protoplasts. Protoplasts were incubated at 30°C in a medium as described in earlier publications (24, 25). The basic incubation medium contained 120 mg of mannitol/ml, 10 mg of glucose/ ml, 20 rmoles of sodium phosphate buffer pH 6.2/ml, 2 pmoles of magnesium chloride/ml, and other additions as described in the legends to the figures and the tables. Lysis of protoplasts and preparation of nuclei. After incubation, the protoplasts were collected by centrifugation and lysed in either one of the following ways. For preparation of a crude 10,OOOg sediment, the protoplasts were resuspended in 10 vol of a cold buffer containing per ml, 20 rmoles of Tris-HCl buffer pH 7.9; 2 pmoles of MgCl,; 2 Hmoles of mercaptoethanol; and 0.2 &moles of EDTA. The lysate was spun for 20 min at 15,000 rpm in the A321 rotor of the International Preparative Ultracentrifuge model R60. Yeast nuclei were prepared as follows. Protoplasts were lysed in lo-20 vol of 0.5 M sucrose in 0.002 M CaCl,, 0.0015 M MgCl,, 0.01 M Tris-HCl buffer pH 7.4, and 0.2% Triton X-190. After five strokes with a tight fitting all glass Potter Homogenizer, the lysate was layered on top of an equal volume of 1 M sucrose in 0.002 mhi CaCl,, 0.0915 mM MgCl,, and 0.05% Triton X-100 and centrifuged for 20 min at 3000 rpm in an International Table top centrifuge. All operations were carried out in a cold room at 2°C. Nuclei were further purified by repeating the centrifugation through 1 M sucrose as described above. Nuclei and 10,OOOg sediment were resuspended in 25% glycerol containing 0.02 M Tris-HCl buffer pH 7.4, 0.002 M MgCl,, 0.002 M mercaptoethanol, and 0.0002 M EDTA (TGMEM). Incubation of nuclei and 10,OOOg sediment. Nuclei and 10,OOOg sediment were incubated with nucleoside triphospbates as follows. To 0.05 ml of a nuclear or 10,OOOg sediment suspension containing 100 pg of protein were added 0.15 ml of 0.01 M MgCl,, and 0.05 ml of a solution containing per ml, 40 pg of unlabeled adenosine triphosphate, guanosine triphosphate, and cytidine triphosphate, 200 wmoles of Tris-HCl buffer pH 7.9, 20 rmoles of mercaptoethanol and 1,5 NCi of 5-Hs-labeled uridine triphosphate. The mixture was incubated for 5 min at 20°C. The reaction was stopped by either the addition of cold five per cent trichloroacetic acid containing 0.02 M sodium pyrophosphate or by the addition of an equal volume of 1 per cent sodium lauryl sulphate in 0.02 M Tris-HCl pH 7.9, 0.0002 M EDTA, and 0.1 M NaCl, when high molecular weight RNA was to be prepared. In the latter case all volumes in the incubation mixture were scaled up four fold. Preparation of high molecular weight RNA. High molecular weight RNA was prepared by phenol de-
324
de KLOET
proteinization of the ncclear lysate in sodium lauryl sulphate obtained as described in the preceding section. The solution was shaken and centrifuged twice with an equal volume of phenol. The RNA was precipitated from the water phase with alcohol as described before (24, 25). Isolation of DNA dependent RNA polymerases. DNA dependent RNA polymerases were isolated by a slight modification of the procedures generally in use. Yeast protoplasts, 10,OOOg sediment or nuclei were resuspended in 20 vol of 0.4 M (NH,),SO, in TGMEM an sonicated for 1 min as described by Adman et al. (19). After centrifugation at 45,000 rpm for 60 min in the A321 rotor of the International Preparative Ultracentrifuge Model B 60, the RNA polymerases were precipitated from the supernatant by saturation with ammonium sulphate. The precipitated enzymes were redissolved in TGMEM and dialyzed against 0.05 M (NH,),SO, in TGMEM. The enzymes were separated by chromatography on DEAE sephadex as described by Adman et al. (19). Estimation of the activity of the isolated RNA polymerases. The activity of the isolated enzymes was tested as follows. The dialyzed enzymes (100 pg of protein) were incubated for 30 min at 30°C in 0.25 ml of a solution containing 20 pg of unlabeled ATP, GTP, and CTP, 5 pmoles of Tris-HCl buffer pH 7.9, 0.5 pmoles of MnCl,, 1.5 pmoles of MgCl,, 4 pmoles of mercaptoethanol, 20 pg of denatured calf thymus DNA and 0.5 &i of Ha-labeled UTP. The reaction was stopped by the addition of 5 ml cold 5% trichloroacetic acid. Density gradient centrifugation of high molecular weight RNA. Aqueous sucrose density gradient centrifugation, using l&33% isokinetic gradients was carried out as described before (24, 25). Alternately sucrose gradients in dimethylsufoxide were used. These were made as described in an earlier paper (31). In the latter case, 5 ml O-10% sucrose gradients in 99% dimethylsulfoxide containing 0.0004 M EDTA and 0.01 M NaCl were used 0.2 ml of the RNA solution in water-dimethylformamide-dimethylsulfoxide (1:1:2 v/v) was layered on top of the gradients and spun for 20 h at 30,000 rpm at 22”C, in the SB 283 rotor of the International Preparative Ultracentrifuge Model B 60. Tubes were punctured, the contents were fractionated and the optical density at 260 nm of the fractions were measured after two fold dilution. The radioactivity of the fractions was determined as described before (24, 25). Separation of messenger RNA from nonmessenger RNA species. Messenger RNA was separated from nonmessenger RNA species by chromatography on cellulose (32, 33) as follows. High molecular weight RNA (200-500 pg) isolated as described before was dissolved in l-ml of distilled water, 9 ml of 0.5 M potassium chloride in 0.01 M Tris pH 7.2 and 0.2 mM MgCl, was added and the sample loaded on a 1 x 10
AND
BELTZ
cm column of Whatman number 1 cellulose equilibrated with the same buffer. After loading, the column was washed with 30 ml of the same KC1 containing buffer. Messenger RNA was eluted with 10 ml of 0.01 M Tris-HCl buffer pH 7.0 in 0.2 mM MgCl,. All operations were carried out at 30°C. Fractions of approximately 2 ml were collected. The optical density of the fractions at 260 nm was estimated and the radioactivity of the fractions was determined after the addition of 100 fig of bovine serum albumin as a carrier and precipitation and washing with cold 5% trichloroacetic acid. When the RNA was to be used for further experimentation, the relevant fractions were pooled, 500 rg of yeast ribosomal RNA added as a carrier and precipitated after the addition of alcohol and sodium acetate as described before. Estimation of protein, DNA, and RNA. Protein was estimated according to Lowry et al. (34). DNA was estimated according to Burton (35), and RNA was estimated from the extinction at 260 nm of a hot tiichloroacetic acid extract, using a value of 25 for the extinction of 1 mg of hydrolyzed RNA per ml and after subtraction of the amount of DNA estimated by the method of Burton. Estimation of the radioactivity of the samples. Samples which had been precipitated with cold 5% trichloroacetic acid were collected on Millipore filters (AAWP), washed three times with cold five per cent trichloroacetic acid and dried in scintillation vials at 85°C for 2 h. The filters were counted in 2.5 ml of a toluene based scintillation solution (4.0 g of Omnifluor, New England Nuclear Coporation) per liter of toluene in a Beckman Liquid Scintillation Counter, Model LS 230. RESULTS
A number of studies have shown that in yeast, like in bacteria, the rate of RNA formation is strongly dependent of the composition of the incubation medium (7-9). In our experiments we have often observed a twenty fold increase in the rate of RNA formation in yeast cells or protoplasts after the addition of 0.3% casamino acids to a nitrogen-free glucose salts medium. In attempts to investigate the mechanism by which amino acid stimulate the formation of RNA in yeast, we have tried to prepare a cell free system that still reflects the activity of the cells it is derived from. In preliminary studies, we fractionated yeast protoplasts simply by lysis in hypotonic medium, followed by differential centrifugation. This procedure yields a heavy cell fraction which actively incorporates
RNA
SYNTHESIS
radioactively labeled nucleoside triphosphates into RNA. Figure la shows that the addition of casamino acids to a protoplast suspension which had been starved for amino acids results in a rapid increase in the rate of incorporation of UTP by the 10,OOOg sediment into cold trichloroacetic acid insoluble material, reflecting the in uiuo increase in capacity for RNA synthesis of the original protoplasts (8) (Fig. lb). Because we are primarily interested in detecting factors in yeast involved in the control of RNA synthesis, we have tried to purify the system further. Undoubtedly, the nuclei or nuclear fragments are responsible for the activity. This can be concluded from the fact that a heavy fraction from the lysate of a mitochondrial DNA less petite mutant of Saccharomyces carlsbergensis shows the same response to a change in incubation conditions as the wild type strain. Furthermore, essentially all RNA polymerase activity of the protoplasts could be recovered from the heavy fraction after sonication in 0.4 M (NH,),SO, according to Adman et al. (19). We have, therefore, tried to prepare nuclei
IN YEAST
NUCLEI
325
according to the methods described by Tonino and Rozijn (36), or by Bhargava and Halvorson (37). Although the first method yielded relatively clean nuclei the difference in activity between nuclei isolated from protoplasts incubated with casamino acids and starved protoplasts was always relatively small and never exceeded a factor two. Later we found that polyvinyl pyrrolidone employed in this method inhibits endogenous RNA polymerase activity of yeast nuclei very strongly, an observation also made by Halvorson and colleagues (32). The method of Bhargava yielded in our hands only very small and variable amounts of nuclei of very low activity. Therefore, we have tried to develop a method for the preparation of relatively clean nuclei which still show a substantial difference in activity for RNA synthesis depending on the original incubation medium for the protoplasts. The method is essentially an adaptation of the procedure described by Lee and Byfield (38), for the isolation of macronuclei of Tetrahymena pyriformis. Based on the DNA content, we have obtained ninety per
FIG. 1. Induction of RNA synthesizing activity of a yeast 10,OOOg sediment after the addition of casamino acids to a protoplast suspension starved for amino acids. Yeast protoplasts (final concentration 1 mg of protein/ml) were incubated for 60 min at 30°C in 30 ml of medium without casamino acids. Casamino acids were added at zero time (final concentration 0.2%) and 4-ml samples were withdrawn at the times indicated and immediately cooled in ice. A lO.OOOg sediment was prepared and incubated with 3H-labeled UTP as described in the text (a). For the determination of the RNA synthesizing capacity of the whole protoplasts (b), 0.5-ml samples were withdrawn and incubated with 10 fig of ‘H-labeled adenine (sp act 0.1 &i per rg) for 5 min at 20°C. The incubation was terminated by the addition of 5 ml of cold 5% trichloroacetic acid. Radioactivity in RNA was estimated as described before (25).
326
de KLOET
cent recovery of the nuclei. The nuclei were judged relatively free of attached cytoplasmic material, although we have never been able to remove all adhering cytoplasmic contaminants. The density of the nuclei was found to be very dependent on the nature of the medium in which the protoplasts had been previously incubated. Upon centrifugation through a stepwise sorbitol gradient, nuclei from starved protoplasts were found to band at about 60% sorbitol, whereas nuclei from protoplasts which had been incubated with casamino acids were much denser and banded between 70 and 80% sorbitol. This indicates that complete separation of the nuclei from other cell constituents which were not or were less influenced by the medium by centrifugation through stepwise sorbitol gradients is not easily attained. The nuclei were found to contain about four per cent of the total RNA of the cell. An analysis of the nuclei prepared by the present method gave an average DNA, RNA, protein ratio of about 1:3:30, in reasonable agreement with the values reported by Tonino et al. and Bhargava et al. (36, 37). These values were again dependent on the medium in which the protoplasts were incubated. Although the protein to DNA ratio of the nuclei did not change substantially, the ratio between DNA-and RNA was-found to fluctuate between 1:4 for nuclei incubated in rich medium against 1:2 nuclei from starved protoplasts. This difference may well be the reason for the difference in density described above. Nuclei isolated by the present method readily incorporate labeled nucleoside triphosphates into RNA. Moreover, such nuclei showed, like the 10,OOOg sediment mentioned before, considerable difference in activity depending on whether they had been prepared from protoplasts which had been starved or incubated with amino acids. In the present paper, we will describe some of the properties of such yeast nuclei, and the RNA which is synthesized. At 3O”C, yeast nuclei synthesize RNA at a rapidly decreasing rate (Fig. 2). The system virtually WaSeS t0 inCOrpOI%tC? labelled UTP after 10 minutes. At 20°C the
AND
BELTZ
system retains its activity considerably longer. The total amount of radioactivity which is incorporated into RNA at 20°C was found to exceed the amount incorporated at 30°C but more important, the RNA synthesized at the lower temperature was found to consist of high molecular weight material sedimenting between 5 and 35s whereas, the RNA synthesized at 30°C consists mainly of low molecular weight material (Fig. 3). Apparently, the RNA synthesized at the lower temperature is also rather stable, for no change in sedimentation profile could be observed when the incubation time was extended from 5-15 min. In attempts to characterize the system further, we have determined the effects of a number of cofactors and inhibitors. The system was found to be dependent on magnesium ions, although the optimum concentration was found to have a rather broad range, between 0.005 M and 0.02 M. Manganese which is indispensable for optimum activity of the isolated eucaryotic DNA dependent RNA polymerases (17, 19) was found to inhibit strongly. Salts of monovalent cations were found to stimulate slightly at low concentrations, at
10
20
30
Tlrnt-~rnllll FIG.
2. Time
course of yeast nuclear RNA syntheat 20” and 30°C. Yeast nuclei were isolated and incubated with nucleoside triphosphates as described under Methods at 20°C (W-----B) and 30°C (04). The total volume of the incubation mixture was 1 ml. Samples (0.1 ml) were withdrawn at the times indicated and the radioactivity incorporated into RNA was estimated as described in the
sizing activity .
text.
RNA
SYNTHESIS
IN YEAST
327
NUCLEI
FIG. 3. Sedimentation pattern of RNA synthesized by isolated yeast nuclei at 20” and 30°C. Nuclei were isolated from protoplasts which had been preincubated with 0.2% casamino acids for 60 min at 30°C. The isolated nuclei were incubated with nucleoside triphosphates for five minutes at 30°C (a) or 20°C (b) as described under Methods. RNA was isolated with phenol SDS and sedimented through sucrose in DMSO.
higher concentrations, (over 0.2 M) the activity of the isolated nuclei was inhibited. Moreover, at increasing concentrations of ammonium sulphate, the difference in activity of nuclei isolated from protoplasts, which were starved or incubated with amino acids, became less and less. The optimal pH of the system was found to be about 7.5. Actinomycin D was found to inhibit very strongly, but (Yamanitin was found to have only marginal effects. These results are summarized in Table I. Based on these results we have taken as optimal conditions for incubation 5 min at 2O”C, 0.006 M MgCl, and no manganese. The RNA synthesized by the nuclei at 20°C was analyzed further for the presence of low molecular weight RNA and polyadenylic acid containing messenger RNA. Figure 4 shows that after prolonged centrifugation of the RNA in sucrose gradients in DMSO, no detectable newly synthesized material sediments in the region of the low molecular weight RNA components indicating that the system is deficient in the formation of low molecular weight RNA species, such as transfer RNA. For the estimation of the capacity of the system to synthesize poly A containing messenger R:NA, we have made use of the fact that such RNA binds to certain types of unmodified cellulose (32, 33). Figure 5 shows that, indeed, a fraction of the RNA (averaging about 15 per cent) binds to cellulose in 0.5 M KCl. Subsequent investigation of the sedimentation properties of
TABLE
I
THE EFFECTS OF SOME MONO- AND DIVALENT CATIONS AND SOME INHIBITORS ON RNA FORMATION BY ISOLATED YEAST NUCLEI” Complete
system
cpm/lOO ccg nuclear protein 3400 5600 7400 7500 7700
+ + + + +
5 x 1O-3 5 x lo-* 2 x
+ + + +
5 x lo-’ M MgCl, + 10m3 M MnCl, 5 x lo-’ M MgCl, + 5 x lOma M MnCI, lo-* M MgCl, + 1Om3 M MnCl, lo-* M MgCl, + 5 x lo-’ M MnCl,
740 520 1530 1140
+ + + +
10m2 M MgCl, lo-’ M MgCl, lo-’ M MgCl, lo-* M MgCl,
8300 8600 9700 6300
+ lo-* + lo-’
lo-’
M MgCl, MgCl, lo-’ MM&l2 M MgCl, lo-* MMgCl, M
M MgCl, M MgCl,
+ + $+
0.05 M KC1 0.1 M KC1 0.2 M KC1 0.4 M KC1
+ actinomycin D (2 Ng) + amanitin (4 rg)
1300 7400
a Yeast nuclei (100 pg of protein) were incubated in 0.3 ml of a solution containing 20 pg of unlabeled ATP, GTP, and CTP, 6 pmoles of Tris-HCI buffer pH 7.5, 0.6 pmoles of mercaptoethanol, 1 PCi of JHlabeled UTP, and other additions as mentioned in the table. Samples were incubated for 5 min at 2O”C, and the cold trichoroacetic acid insoluble radioactivity was estimated.
this RNA fraction showed that it sediments at about 14S, close to the value found earlier for yeast messenger RNA synthesized in uiuo (49) (Fig. 6). A further study
328
de KLOET
AND BELT2
In order to study the initiation of RNA synthesis, we have investigated the incorporation of -r32P-labeled adenosine and guanosine triphosphate. The latter nucleotides were the predominant initiating nucleotides in RNA formation by isolated yeast DNA dependent RNA polymerases and we have assumed that the same is the case in isolated nuclei or in uiuo although no proof exists (40). The results of these experiments showed that most of the label Fract,on number that was incorporated into material which FIG. 4. Sedimentation pattern of RNA synthesized by isolated yeast nuclei. Yeast nuclei were remains in the water phase after shaking incubated as described under Fig. 3 and the isolated with phenol is largely insensitive to RNARNA was centrifuged through a sucrose gradient in ase, indicating that it represents a contamDMSO containing 0.01 M KC1 for 30 h at 30,000 rpm. inant. To characterize the incorporated radioactivity further, we have hydrolyzed the RNA in alkali and subjected the hydrolysate to paper chromatography. No label was found to chromatograph in the region where nucleoside tetraphosphates are expected to appear, using nucleoside triphosphates as markers upon chromatography in 75 per cent alcohol according to Lane (41) or in 0.1 M phosphate according to Cashel (42). Finally upon sedimentation through a sucrose gradient, virtually all the labeled material remained in the upper half of the gradient whereas the newly synthesized mainly between 10 and FIG. 5. Fractionation of RNA synthesized by iso- RNA sediments 35s (Fig. 3). These observations indicate to lated yeast nuclei on a cellulose column. Yeast nuclei were incubated with ‘H-labeled UTP at 20°C as us that the mechanism for initiation of described before. The RNA was isolated and fractionRNA synthesis in yeast nuclei is severely
i I 05/
ated by chromatography Feigelson (32).
on cellulose as described by
I
0.8 c -1 80 of the effect of cy-amanitin showed that the amount of polyadenylic acid containing messenger RNA was not affected, even not at concentrations as high as 25 &ml. The data described above show that isolated yeast nuclei can elongate RNA molecules of considerable molecular weight and can complete messenger RNA molecules since polyadenylic acid, which causes messenger RNA to adhere to cellulose, is added posttranscriptionally (39). FIG. 6. Sedimentation pattern of yeast messenger We were therefore interested in whether RNA synthesized by isolated nuclei and fractionated yeast nuclei prepared by the present on cellulose RNA synthesized by isolated yeast nuclei method could carry out other important was fractionated as described under Fig. 5. The steps in RNA formation, like initiation and material eluting with buffer containing no KC1 was processing, e.g., of ribosomal precursor collected and centrifuged through a dimethylsulfoxide RNA. sucrose gradient.
RNA
SYNTHESIS
impaired, although we cannot exclude that in yeast nuclei terminal phosphates of the initiating nucleotides have a very short half life which makes it impossible to detect such phosphate residues by the present techniques. The conclusion that isolated nuclei cannot initiate RNA synthesis is in agreement with the conclusion reached by other investigators for other eucaryotic nuclei (43, 44). Processing of ribosomal RNA precursors was studied by pulse labeling in uiuo followed by a continued incubation of the labeled nuclei. in uitro. The data in Fig. 7 show that no detectable processing of ribosomal precursor RNA occurs under the present experimental conditions, both when 8-3H-labeled adenosine or [Me3H]methionine are used as labeled precursors. The continuous drop in RNA synthesizing capacity of the nuclei during incubation at 20°C or 30°C might, therefore, well reflect the inability of the nuclei to initiate RNA sythesis coupled to a continued elongation and termination of the formation of RNA molecules. Indeed, as Table II shows, the rate at which the nuclei loose their capacity of RNA synthesis is reduced at a higher rate when incubated in the presence of nucleoside triphosphates than when incubated in the absence. This might be
t0
20
10 Froctlon
20
number
FIG. 7. Stability of preformed RNA in nuclei upon reincubation in vitro. Yeast protoplasts were preincubated in casamino acids containing medium for 60 min at 30°C and subsequently labeled for 2 min with 8-sH-labeled adenine. Nuclei were isolated and reincubated for 10 min at 20°C with unlabeled triphosphates as described in the text, replacing the labeled UTP by 20 pg of the unlabeled compound. RNA was isolated and centrifuged through sucrose DMSO as described. (a) Pattern before reincubation; (b) pattern after reincubation.
IN YEAST
329
NUCLEI
taken as a further indication that lack of capacity to initiate RNA synthesis is the cause of this reduction of the activity of the nuclei. A further analysis showed however that such an incubation of nuclei with nucleoside triphosphates also reduces the activity of the isolated RNA polymerases very strongly, demonstrating that lack of capacity to initiate RNA synthesis as well as inactivation of RNA polymerase causes the drop in activity of the nuclei (Table II). The RNA synthesizing activity of the nuclei, like the 10,OOOg sediment, increases very rapidly when protoplasts which had been starved for amino acids are reincubated in a medium containing amino acids. The increase is completely inhibited by cycloheximide. In this respect, the increase in activity resembles the formation of an induced enzyme. The increase in activity of RNA synthesizing capacity of the nuclei might be due to an increase in activity of the RNA polymerizing enzymes TABLE Loss AFTER
OF RNA INCUBA~ON
II
SYNTHESIZING CAPACITY IN NUCLEI WITH NUCLEOSIDE TRIPHOSPHATES’
cpm/lOO Pg of nuclear protein (a) (b) (c) (d) (e) (f)
Nuclei not preincubated Nuclei preincubated for 10 min Nuclei preincubated without NTP’s Isolated RNA polymerase from (a) Isolated RNA polymerase from (b) Isolated RNA polymerase from (c)
940 430 2370 370 1550
“Yeast nuclei (100 rg of protein) were preincubated at 20°C in 0.25 ml of a solution containing 20 pg of ATP, GTP, and CTP and 1 pg of unlabeled UTP, 6 pmoles of Tris-HCl buffer pH 7.9, 0.6 pmoles of mercaptoethanol, 3 pmoles of MgCl, under conditions as indicated in the table. For the estimation of the activity of the nuclei 0.05 ml of a solution containing 20 pg of unlabeled ATP, GTP, and CTP and 1 bg of “H-labeled UTP (sp act 3 &i/pg) was added after 10 min of preincubation and the incubation continued for another 10 min. For the isolation of the RNA polymerases the amounts of material in the experiments were scaled up tenfold and the enzymes isolated by sonication and precipitation with ammonium sulphate as described under Methods. The activity of the enzyme was tested after dialysis as described in the text.
330
de KLOET
as well as to an increase of the actual amount of the enzymes. Figure 8 shows that two DNA dependent RNA polymerases can be readily extracted from nuclei prepared with Triton X 100. The data on the effect of cy-amanitin show that the elution pattern upon chromatography on DEAE sephadex is similar to the pattern observed by others (19-21) with two components (AI and AII) which are not sensitive to the antibiotic, and a major components B which is sensitive to cy-amanitin. A fourth component eluting after B, also a-amanitin insensitive was infrequently observed, which might be just the result of the concentration dependence of the activity of this enzyme (45). Table III shows that when the RNA polymerases are isolated, the total activity of the enzymes, as well as the ratio between cu-amanitin resistant and sensitive enzymes remains unchanged demonstrating that only the activity of the RNA polymerases is affected as a result of the change in incubation conditions, a conclusion similar to that reached by Gross and Pogo (28) in their studies on RNA synthesis by nuclei of S. cerevisiae.
FIG. 8. Elution pattern of RNA polymerase isolated from yeast upon chromatography on DEAE Sephadex. RNA polymerizing enzymes were isolated and fractionated as described under Methods. The activity of the fractions of the eluate was estimated by incubating 0.05-ml samples for 30 min at 30°C with 0.2 ml of a solution containing 20 rg of unlabeled ATP, CTP, and GTP, 5 rmoles of Tris-HCl buffer pH 7.9, 0.5 nmoles of MnCl,, 1.5 rmoles of MgCI,, 4 rmoles of mercaptoethanol, 20 pg of denatured calf thymus DNA and 0.5 &i of Ha-labeled UTP. The reaction was stopped by the addition of 5 ml cold 5% trichloroacetic acid.
AND
BELTZ TABLE
III
ACTNITY OF ISOLATED RNA POLYMERASES ISOLATED FROM YEAST NUCLEI PREPARED FROM PROTOPLA~TS WHICH HAD BEEN STARVED OR INCUBATED WITH AMINO ACIDS” CPm/loo CcfT of nuclear protein Nuclei Nuclei with
from starved protoplasts from protoplasts incubated casamino acids
RNA polymerases isolated starved protoplasts +a-amanitin (2rg)
from
RNA polymerases from protoplasts incubated with amino acids +cu-amanitin (2 ng)
1470 11700
8317 5637 8737 5314
’ Yeast nuclei were isolated from protoplasts which had been either starved for 60 min by incubation in the absence of casamino acids or which had been incubated in basic medium with the addition of 0.2% casamino acids. The RNA synthesizing capacity of the nuclei was tested as described under Methods. The isolation of the RNA polymerase activity and the estimation of the enzymatic activity has been described in the text.
A change in activity of the RNA polymerizing activity of the nucleus may be the result of a change in the rate at which individual enzyme molecules catalyze the formation of RNA molecules. It may also be that under conditions of amino acid starvation part of the polymerase is unavailable for transcription of endogenous DNA, and possibly available for the transcription of added foreign DNA. The latter possibility was investigated. As was shown above manganese ions inhibit the endogenous RNA synthesizing activity of the nuclei, although the isolated RNA polymerases depend heavily on the presence of this ion for optimal activity (Ii’, 19). Table IV shows that the nuclei from starved protoplasts show a more substantial increase in activity upon the addition of denatured calf thymus DNA compared to nuclei isolated from protoplasts incubated in a rich medium. The latter result suggest that activation of latent RNA polymerase is at least in part, responsible for
RNA SYNTHESIS TABLE
IV
THE EFFECT OF THE ADDITION OF DNA ON RNA SYNTHESIS BY ISOLATED YEAST NUCLEP
cpm/lOO I.%of nuclear protein
Nuclei from protoplasts incubated without casamino acids + 20 pg of denatured calf thymus DNA Nuclei from protoplasts incubated with casamino acids + 2Opg of denatured calf thymus DNA
990 2770 5600 5100
“Yeast protoplasts (1 mg of protein/ml) were preincubated at 30” in lo-ml medium without casamino acids. After 60 min casamino acids (final concentration 0.2%) were added and the incubation continued for another 30 min. Nuclei were isolated and incubated for 5 min at 20°C in 0.3 ml of a medium containing 20 rg of unlabeled ATP, GTP, and CTP, 6 rmoles of Tris-HCl buffer pH 7.9, 0.6 rmoles of mercaptoethanol, 3 pmoles of MgCl,, 0.6 rmoles of MnCl,, 1 PCi of [3H]UTP and denatured calf thymus DNA as indicated.
the increase in RNA synthesis upon the addition of amino acids to the medium. In yeast, like in other microorganisms, not only the rate of RNA formation is subject to environmental control, but also the ratio in which ribosomal, messenger and transfer RNA are formed. Messenger RNA is in general relatively less influenced than ribosomal RNA formation. A similar result is obtained when the composition of the RNA synthesized by the isolated nuclei
Frocfvsn
IN YEAST NUCLEI
331
is studied. Upon analysis by cellulose chromatography, it was found that RNA synthesized in nuclei isolated from yeast protoplasts which had been incubated in the presence of amino acids contains relatively less polyadenylic acid containing messenger RNA than RNA synthesized by nuclei isolated from starved protoplasts. (15 vs 30%). The data in Fig. 9 show that the former RNA contains more material sedimenting ahead of ribosomal RNA in a dimethylsulfoxide sucrose gradient, which is the region of the ribosomal precursor RNA. Therefore, although the nuclei as isolated by the present procedure are certainly deficient in a number of aspects of RNA synthesis, the composition of the synthesized RNA reflects still the activity of the original protoplasts for the preparation of the nuclei. DISCUSSION
The present study shows that isolated yeast nuclei continue to synthesize RNA for a short period. The observation that more RNA is made and that it has a higher molecular weight when the nuclei are incubated at a low temperature (20°C) than when it is synthesized at a higher temperature (30°C) resembles the observations made by Marzluff et al. (14) in their studies on RNA synthesis by mouse myeloma nuclei. Again, like has been found previously by Tata and others (43), manganese ions inhibit nuclear RNA synthesis although this
number
FIG. 9. Sedimentation pattern of RNA synthesized by yeast nuclei. Yeast nuclei were isolated from protoplasts incubated with or without amino acids and incubated for 5 min at 20°C as described under Methods. RNA was isolated and centrifuged through sucrose in DMSO as described. (a) Sedimentation pattern of RNA synthesized by nuclei isolated from protoplasts starved for amino acids. (b) Sedimentation pattern of RNA synthesized by nuclei isolated from protoplasts incubated with amino acids.
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de KLOET
divalent cation is necessary for the activity of the isolated enzymes (17, 19-21). Contrary to what has been found for nuclei of other organisms, we have never observed any substantial effect of a-amanitin on RNA synthesis in yeast nuclei, although actinomycin D inhibits very strongly. Although it cannot be ruled out that (Yamanitin cannot penetrate yeast nuclei under the present conditions, another explanation might be that in yeast nuclei messenger RNA as well as the other RNA species are made by a-amanitin resistant enzymes. A somewhat similar but not so extreme conclusion has been reached by Zylber and Penman (15) who concluded that heterogeneous nuclear RNA in HeLa cells is synthesized by two distinct enzymes, one sensitive, one insensitive to the antibiotic. These results are somewhat different from those obtained by Gross and Pogo (28) in their studies on nuclei of S. cereuisiae. These authors observed a substantial effect of cr-amanitin. In their experiments mangenese was included in the incubation medium and the incubation time was substantially longer (30 min) than in the present study. In our studies we have found however that manganese inhibits the activity of the nuclei and consequently we have omitted it from our incubation medium, in order to maintain optimum conditions for RNA formation. Compared to nuclei isolated by others from different organisms (13, 45, 46), yeast nuclei show differences and similarities with respect to a number of important aspects of the biosynthesis of RNA. Based on our experiments with y-labeled [“‘PInucleoside triphosphates yeast nuclei do not seem to initiate the synthesis of new RNA molecules, although the possibility that the terminal phosphate is cleaved off shortly after initiation makes this conclusion at least tentative. The conclusion is however in agreement with that reached by others in their studies of RNA synthesis in isolated mammalian nuclei (45, 44). The decrease in RNA synthesis with time cannot only be ascribed to a failure of the mechanism of initiation, since we have also observed that the activity of the isolated RNA synthesizing enzymes drops
AND
BELTZ
sharply during incubation of the nuclei with nucleoside triphosphates. Earlier studies (8, 9, 48) had shown that in uiuo the ribosomal precursor in yeast sediments as material of about 38S, whereas messenger RNA sediments with an average sedimentation coefficient of 14s (49). Based on the sedimentation data and the chromatographic properties on cellulose, both ribosomal precursor RNA and messenger RNA are synthesized by isolated yeast nuclei. No low molecular weight RNA has been found among the products. Whether this means that transfer RNA is synthesized in yeast as part of a large precursor which is not processed in the course of the experiment or whether the system is actually deficient in the transcription of this RNA component cannot be concluded from the present data. No processing of ribosomal precursor RNA has been shown to occur in isolated yeast nuclei, although isolated nuclei from amplibia (13, 50-52) and certain insects (53) can carry out a number of steps required for the maturation of ribosomal RNA. A major factor in the regulation of the formation of ribosomal RNA in eucaryotes operates through control of the maturation process, which is closely connected to the ability of the cell to carry out protein synthesis. Most likely this control is indirect and related to the availability of certain proteins. In yeast nuclei such a pool might be smaller than in other organisms. On the other hand, the in vitro processing of ribosomal precursor RNA by isolated nucleolar endonuclease (48) has been shown to be strongly dependent on the concentration of divalent cations, so that our incubation conditions might be far from ideal to observe any processing. The finding that labeled messenger RNA can be isolated by cellulose chromatography shows that the nuclei can complete the synthesis of at least this RNA species, since the polyadenylic acid moiety is added post transcriptionally (39). The present study shows that the higher rate of RNA synthesis in the nuclei isolated from protoplasm incubated in the presence of amino acids as compared to the rate in nuclei isolated from starved protoplasts
RNA
SYNTHESIS
can at least in part be explained as being the result of the binding of more RNA polymerase in a form which makes it unavailable for the transcription of added template, and which allows it to transcribe only endogenous DNA. Whether this is accompanied by any additional structural change or change of protein content and composition of the nuclei is at present under investigation. ACKNOWLEDGMENTS This work was supported by American Cancer Society and National Science Foundation. debted to Dr. T. Wieland for a-amanitin.
Grants NP 80 of the NB Pi B 2200 of the The authors are inthe generous gift of
REFERENCES 1. RYAN, J. L., AND BOREK, E. (1971) Progress in Nucleic Acid Research and Molecular Biology 11, 193. 2. PACE, N. R. (1973) Bacterial. Reu. 37, 562. 3. HALL, B., AND GALLANT, J. (1972) Nature New Biol. 237, 131. 4. CASHEL, M., ,~ND GALLANT, J. (1969) Nature (London) 221, 838. 5. CASHEL, M., AND KAHLBACHER, B. (1970) J. Biol. Chem. 245, 2309. 6. ALBERGHINA, F. A. M., SCHIAFONATI, L., ZARDI, L., AND STURANI, E. (1973) Biochim. Biophys. Acta 312, 435. 7. ROTH, R. M., XND DAMPIER, C. (1972) J. Bacterial. 109, 773. 8. DE KLOET, S. R. (1966) Biochem. J. 99, 566. 9. RETEL, J., AND PLANTA, R. J. (1967) Eur. J. Biochem. 3, 248. 10. CHAMBERLIN, M. (1969) Cold Spring Harbor Symp. Quont. Biol. 35, 851. 11. PETTIJOHN, D. E. (1973) Nature New Biol. 234, 204. 12. TRAVERS, A., AND BUCKLAND, R. (1973) Nature New Biol. 243, 257. 13. REEDER, R. H., AND ROEDER, R. G. (1972) J. Mol. Biol. 67, 483. 14. MARZLUFF, W. F., MURPHY, E. C., AND HUANG, R. C. C. (1973) Biochemistry 12, 3440. 15. ZYLBER, E. A., AND PENMAN, S. (1971) Proc. Nat. Acad. Sci. USA 68, 2861. 16. ROEDER, R. G., AND RUITER, W. J. (1970) Proc. Nat. Acad. Sci. USA 65, 675. 17. BLAITI, S. P., INGELS, C. Y., LIADELL, T. Y., MORRIS, P. W., WEAVER, R. F., WEISBERG, F., AND Rumit, W. J. (1970) Cold Spring Harbor Symp. Quant. Biol. 35, 649. 18. HILDEBRANDT, A. H., SEBASTIAN, J., AND HALVORSON., H. 0. (1973) Nature New Biol. 246, 74.
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19. ADMAN, R., SCHULTZ, L. D., AND HALL B. D. (1972) Proc. Nat. Acad. Sci. USA 69, 1703. 20. PONTA, H., PONTA, U., AND WINTERSBERGER, E. (1972) Eur. J. Biochem. 29, 110. 21. BROGT, TK M., ANDPLANTA, R. J. (1972) Fed. Eur. Biochem. Sot. Lett. 26, 47. 22. WILLEMS, M., PENMAN, M., AND PENMAN, S. (1967) J. Cell. Biol. 41, 177. 23. WAROQUIER, R., AND SCHERRER, K. (1969) Eur. J. Biochem. 10, 362. 24. MAYO, V. S., ANDREAN, B. A. G., AND DE KLOET, S. R. (1968) Biochim. Biophys. Acta 169,297. 25. MAYO, V. S., AND DE KLOET, S. R. (1972) Arch. Biochem. Biophys. 153,508. 26. DE KLOET, unpublished results. 27. SAJDEL-SULKOSWKA, E. M., BHARGAVA, M. M., ARNAUD, M. V., AND HALVORSON, H. O., (1973) Biochem. Biophys. Res. Comm. 56, 496. 28. GROSS, K. J., AND Poco, A. 0. (1974) J. Biol. Chem. 249, 568. 29. WINTERSBERGER, U., SMITH, P., AND LETNANSKY, K. (1973) Eur. J. Biochem. 33, 123. 30. DE KLOET, S. R., VAN WERMESKERKEN, R. K. A., AND KONINGSBERGER, V. V. (1961) Biochim. Biophys. Acta, 47, 138. 31. DE KLOET, S. R., MAYO, V. S., AND ANDREAN, B. A. G. (1974) Arch. Biochem. Biophys. 162, 313-315. 32. SCHULTZ, G., BEATO, M., AND FEIGELSON, P. (1972) Biochem. Biophys. Res. Comm. 49,680. 33. SULLIVAN, N., AND ROBERTS, K. W. (1973) Biochemistry 12, 2395. N. J., FARR, A. L., 34. LOWRY, 0. H., ROSEBROUGH, AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265. 35. BURTON, K. (1952) Biochem. J. 62, 315. 36. ROZIJN, T. H., AND TONINO, G. J. M. (1964) Biochim. Biophys. Acta 91, 105. 37. BHAKGAVA, M. M., AND HALVORSON, H. 0. (1971) J. Ceil Biol. 49, 423. 38. LEE, Y. C., AND BYFIELD, J. E. (1970) Biochemistry 9, 3947. Reu. Biochem. 39. WEINBERGER, R. A. (1973) Annu. 42, 329. 40. FREDERICK, E. W., MAITRA, U., AND H~RWITZ, J. (1969) J. Biol. Chem. 244, 413. 41. LANE, B. G. (1963) B&him. Biophys. Acta 72, 110. 42. CASHEL, M., LAZZARINI, R. A., AND KAHLBACHER, B. (1969) J. Chromatogr. 40, 103. 43. POGO, A. (1969) Biochim. Biophys. Acta 182, 57. 44. POGO, A. O., ALLFREY, V. G., AND MIRSKY, A. E. (1966) Proc. Nat. Acad. Sci. USA 56, 550. 45. HALL, B. D. Personal communication. 46. JACOB, S. T., SAJDEL, E. M., AND MUNRO, H. N. (1968) Biochem. Biophys. Res. Comm. 32,831. 47. WIDNELL, C. C., AND TATA, S. R. (1966) Biochim. Biophys. Acta 123, 478.
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48. UDEM,
S. A., AND WARNER,
J. R. (1972)
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Bid. 65, 227. V. S., AND ANDREAN,
B. A.
Biophys. Res. Comm. 40,
454. Biol. 67, 433.
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51. CASTON,
J. D.,
AND JONES,
P. H. (1972)
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Mol.
Biol. 69, 19.
49. DE KLOET, S. R., MAYO, G. (1970) B&hem. 50. REEDER, R. H.,
AND
AND BROWN,
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52. KELLEY,
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OF BlOCHEMIS’IRY
Control
AND
BIOPHYSICS
of the Formation
Ribonucleic
of
(1975)
of Ribonucleic
Acid in Yeast: Synthesis
Acid in a Nuclear Fraction carlsbergensis
SIWG Department
167, 322-334
R.
Biological
DE
KLOET
Science,
WILLIAM
AND
Florida Received
State May
University,
of
of Saccharomyces
R. BELTZ Tallahassee,
Florida
32306
23, 1974
A study has been made of the nature of the RNA synthesized by isolated yeast nuclei. At 20°C the product consists of high molecular weight RNA sedimenting between 10 and 3OS, including polyadenylic acid containing messenger RNA. At 30°C the synthesized material is mainly of low molecular weight. Optimal conditions for synthesis include the presence of magnesium ions (0.01 M). Manganese ions were found to inhibit very strongly. Nuclei of yeast protoplasts incubated in the presence of amino acids were found to be up to ten times more active than nuclei isolated from starved protoplasts. Yet the activity of the isolated enzymes remained unchanged demonstrating that the activity instead of the actual amount of the enzymes was altered.
The mechanism by which the formation of ribonucleic acid, in particular ribosomal ribonucleic acid is regulated has been extensively investigated in procaryotes, especially Escherichia coli. Genetic analysis has shown that the formation of ribosomal RNA in this organism is under the influence of the RC gene, which in the wild type state correlates the rate of synthesis of ribosomal RNA with the availability of a required amino acid (1, 2). Recent investigations have shown that the RC gene affects the translational apparatus in E. coli (3, 4) and controls the level of a nucleotide 3’-diphosphoguanosine-5’diphosphate (ppGpp)’ (4, 5). The actual involvement of this nucleotide in the biosynthesis of RNA is however still under investigation. No such investigation has been possible in eucaryotes, no RC gene has been identified and the presence of ppGpp has not been reported (6). Yet eucaryotes like yeast show a definite correlation between the ’ Abbreviations used: 3’-diphosphoguanosine-5’diphosphate, ppGpp; 0.02 Tris-HCl buffer pH 7.4, 0.002 M MgCl,, 0.002 M mercaptoethanol, 0.0002 M EDTA, TGMEM; and dimethylsulfoxide, DMSO. 322 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
rate of RNA synthesis and the nature of the growth medium (7, 8, 9), and the addition of amino acids to a minimal medium or the addition of a required amino acid results in a dramatic increase in the rate at which ribosomal RNA is formed. A fundamental difference between procaryotes and eucaryotes is found in the nature of the enzymes which transcribe DNA into RNA. In E. coli a single DNA dependent RNA polymerase occurs which is responsible for the formation of messenger as well as ribosomal RNA (10, 11, 12). In eucaryotes the formation of RNA is carried out by several enzymes which show a distinct nuclear localization and which differ in a number of biochemical aspects. Since one of these enzymes is found in the nucleolus, this enzyme is most likely responsible for the formation of ribosomal RNA, whereas the one or more enzymes found in the nucleoplasm are probably responsible for the transcription of messenger sequences (13-18). Although it has not been possible to fractionate yeast nuclei into similar nucleolar and nucleoplasmic fractions, the similarity in elution patterns of the enzymes upon DEAE Sephadex
RNA
SYNTHESIS
chromatography as compared to the profile obtained from other eucaryotes with more easily identifiable nuclear subfractions (19, 20, 21) suggests strongly that yeast ribosomal and messenger RNA formation is also carried out by different enzymes, very similar to those in higher eucaryotes. The presence of different enzymes for the formation of ribosomal and other RNA species makes an independent control mechanism for the formation of ribosomal RNA very feasible. On the other hand many data (22, 23) suggest that a major regulatory step in the formation of at least ribosomal RNA occurs through the regulation of the maturation process. Inhibition of the formation of ribosomal RNA in yeast by a number of different agents like cycloheximide (8, 24), 5-fluorouracil (25), and heat (26) all give rise to the accumulation of precursor stages of ribosomal RNA. A major prerequisite for the understanding of the mechanism by which the formation of ribosomal RNA is regulated in procaryotes and eucaryotes is the preparation of a cell free system in which this process can be adequately studied. It is the intention of this paper to describe some of the characteristics of a cell free system derived from yeast protoplasts which still reflects the RNA synthesizing capacity of the whole protoplasts from which it has been derived. Several papers have been published dealing with this subject (27, 28, 29). In this paper we will deal especially with the nature of the RNA synthesized in this system as well as with some of the aspects of the mechanism by which the formation of RNA is controlled in yeast. MATERIALS
AND
METHODS
Strains, Saccharomyces carlsbergensis (N.C.T.C. 74) was used for this investigation. Growth of the organism as well as the conversion of logarithmic cells into protoplasm with snail enzyme has been described before (30). Chemicals. [5--*H]Uridine triphosphate (sp act 15 Wmmole), [8-3H]guanosine triphosphate (sp act 20 Ci/mmole) were purchased from Schwarz-Mann, Orangeburg, NJ. y-32P-labeled adenosine triphosphate, (sp act 68 Ci/mmole) r-32P-labeled guanosine triphosphate (sp act 52 Ci/mmole) and gSH-labeled adenine (sp act 20 Ci/mmole) were products of I.C.N.
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All other chemicals were the regular commercially available brands, highest grade of purity. Incubation of protoplasts. Protoplasts were incubated at 30°C in a medium as described in earlier publications (24, 25). The basic incubation medium contained 120 mg of mannitol/ml, 10 mg of glucose/ ml, 20 rmoles of sodium phosphate buffer pH 6.2/ml, 2 pmoles of magnesium chloride/ml, and other additions as described in the legends to the figures and the tables. Lysis of protoplasts and preparation of nuclei. After incubation, the protoplasts were collected by centrifugation and lysed in either one of the following ways. For preparation of a crude 10,OOOg sediment, the protoplasts were resuspended in 10 vol of a cold buffer containing per ml, 20 rmoles of Tris-HCl buffer pH 7.9; 2 pmoles of MgCl,; 2 Hmoles of mercaptoethanol; and 0.2 &moles of EDTA. The lysate was spun for 20 min at 15,000 rpm in the A321 rotor of the International Preparative Ultracentrifuge model R60. Yeast nuclei were prepared as follows. Protoplasts were lysed in lo-20 vol of 0.5 M sucrose in 0.002 M CaCl,, 0.0015 M MgCl,, 0.01 M Tris-HCl buffer pH 7.4, and 0.2% Triton X-190. After five strokes with a tight fitting all glass Potter Homogenizer, the lysate was layered on top of an equal volume of 1 M sucrose in 0.002 mhi CaCl,, 0.0915 mM MgCl,, and 0.05% Triton X-100 and centrifuged for 20 min at 3000 rpm in an International Table top centrifuge. All operations were carried out in a cold room at 2°C. Nuclei were further purified by repeating the centrifugation through 1 M sucrose as described above. Nuclei and 10,OOOg sediment were resuspended in 25% glycerol containing 0.02 M Tris-HCl buffer pH 7.4, 0.002 M MgCl,, 0.002 M mercaptoethanol, and 0.0002 M EDTA (TGMEM). Incubation of nuclei and 10,OOOg sediment. Nuclei and 10,OOOg sediment were incubated with nucleoside triphospbates as follows. To 0.05 ml of a nuclear or 10,OOOg sediment suspension containing 100 pg of protein were added 0.15 ml of 0.01 M MgCl,, and 0.05 ml of a solution containing per ml, 40 pg of unlabeled adenosine triphosphate, guanosine triphosphate, and cytidine triphosphate, 200 wmoles of Tris-HCl buffer pH 7.9, 20 rmoles of mercaptoethanol and 1,5 NCi of 5-Hs-labeled uridine triphosphate. The mixture was incubated for 5 min at 20°C. The reaction was stopped by either the addition of cold five per cent trichloroacetic acid containing 0.02 M sodium pyrophosphate or by the addition of an equal volume of 1 per cent sodium lauryl sulphate in 0.02 M Tris-HCl pH 7.9, 0.0002 M EDTA, and 0.1 M NaCl, when high molecular weight RNA was to be prepared. In the latter case all volumes in the incubation mixture were scaled up four fold. Preparation of high molecular weight RNA. High molecular weight RNA was prepared by phenol de-
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proteinization of the ncclear lysate in sodium lauryl sulphate obtained as described in the preceding section. The solution was shaken and centrifuged twice with an equal volume of phenol. The RNA was precipitated from the water phase with alcohol as described before (24, 25). Isolation of DNA dependent RNA polymerases. DNA dependent RNA polymerases were isolated by a slight modification of the procedures generally in use. Yeast protoplasts, 10,OOOg sediment or nuclei were resuspended in 20 vol of 0.4 M (NH,),SO, in TGMEM an sonicated for 1 min as described by Adman et al. (19). After centrifugation at 45,000 rpm for 60 min in the A321 rotor of the International Preparative Ultracentrifuge Model B 60, the RNA polymerases were precipitated from the supernatant by saturation with ammonium sulphate. The precipitated enzymes were redissolved in TGMEM and dialyzed against 0.05 M (NH,),SO, in TGMEM. The enzymes were separated by chromatography on DEAE sephadex as described by Adman et al. (19). Estimation of the activity of the isolated RNA polymerases. The activity of the isolated enzymes was tested as follows. The dialyzed enzymes (100 pg of protein) were incubated for 30 min at 30°C in 0.25 ml of a solution containing 20 pg of unlabeled ATP, GTP, and CTP, 5 pmoles of Tris-HCl buffer pH 7.9, 0.5 pmoles of MnCl,, 1.5 pmoles of MgCl,, 4 pmoles of mercaptoethanol, 20 pg of denatured calf thymus DNA and 0.5 &i of Ha-labeled UTP. The reaction was stopped by the addition of 5 ml cold 5% trichloroacetic acid. Density gradient centrifugation of high molecular weight RNA. Aqueous sucrose density gradient centrifugation, using l&33% isokinetic gradients was carried out as described before (24, 25). Alternately sucrose gradients in dimethylsufoxide were used. These were made as described in an earlier paper (31). In the latter case, 5 ml O-10% sucrose gradients in 99% dimethylsulfoxide containing 0.0004 M EDTA and 0.01 M NaCl were used 0.2 ml of the RNA solution in water-dimethylformamide-dimethylsulfoxide (1:1:2 v/v) was layered on top of the gradients and spun for 20 h at 30,000 rpm at 22”C, in the SB 283 rotor of the International Preparative Ultracentrifuge Model B 60. Tubes were punctured, the contents were fractionated and the optical density at 260 nm of the fractions were measured after two fold dilution. The radioactivity of the fractions was determined as described before (24, 25). Separation of messenger RNA from nonmessenger RNA species. Messenger RNA was separated from nonmessenger RNA species by chromatography on cellulose (32, 33) as follows. High molecular weight RNA (200-500 pg) isolated as described before was dissolved in l-ml of distilled water, 9 ml of 0.5 M potassium chloride in 0.01 M Tris pH 7.2 and 0.2 mM MgCl, was added and the sample loaded on a 1 x 10
AND
BELTZ
cm column of Whatman number 1 cellulose equilibrated with the same buffer. After loading, the column was washed with 30 ml of the same KC1 containing buffer. Messenger RNA was eluted with 10 ml of 0.01 M Tris-HCl buffer pH 7.0 in 0.2 mM MgCl,. All operations were carried out at 30°C. Fractions of approximately 2 ml were collected. The optical density of the fractions at 260 nm was estimated and the radioactivity of the fractions was determined after the addition of 100 fig of bovine serum albumin as a carrier and precipitation and washing with cold 5% trichloroacetic acid. When the RNA was to be used for further experimentation, the relevant fractions were pooled, 500 rg of yeast ribosomal RNA added as a carrier and precipitated after the addition of alcohol and sodium acetate as described before. Estimation of protein, DNA, and RNA. Protein was estimated according to Lowry et al. (34). DNA was estimated according to Burton (35), and RNA was estimated from the extinction at 260 nm of a hot tiichloroacetic acid extract, using a value of 25 for the extinction of 1 mg of hydrolyzed RNA per ml and after subtraction of the amount of DNA estimated by the method of Burton. Estimation of the radioactivity of the samples. Samples which had been precipitated with cold 5% trichloroacetic acid were collected on Millipore filters (AAWP), washed three times with cold five per cent trichloroacetic acid and dried in scintillation vials at 85°C for 2 h. The filters were counted in 2.5 ml of a toluene based scintillation solution (4.0 g of Omnifluor, New England Nuclear Coporation) per liter of toluene in a Beckman Liquid Scintillation Counter, Model LS 230. RESULTS
A number of studies have shown that in yeast, like in bacteria, the rate of RNA formation is strongly dependent of the composition of the incubation medium (7-9). In our experiments we have often observed a twenty fold increase in the rate of RNA formation in yeast cells or protoplasts after the addition of 0.3% casamino acids to a nitrogen-free glucose salts medium. In attempts to investigate the mechanism by which amino acid stimulate the formation of RNA in yeast, we have tried to prepare a cell free system that still reflects the activity of the cells it is derived from. In preliminary studies, we fractionated yeast protoplasts simply by lysis in hypotonic medium, followed by differential centrifugation. This procedure yields a heavy cell fraction which actively incorporates
RNA
SYNTHESIS
radioactively labeled nucleoside triphosphates into RNA. Figure la shows that the addition of casamino acids to a protoplast suspension which had been starved for amino acids results in a rapid increase in the rate of incorporation of UTP by the 10,OOOg sediment into cold trichloroacetic acid insoluble material, reflecting the in uiuo increase in capacity for RNA synthesis of the original protoplasts (8) (Fig. lb). Because we are primarily interested in detecting factors in yeast involved in the control of RNA synthesis, we have tried to purify the system further. Undoubtedly, the nuclei or nuclear fragments are responsible for the activity. This can be concluded from the fact that a heavy fraction from the lysate of a mitochondrial DNA less petite mutant of Saccharomyces carlsbergensis shows the same response to a change in incubation conditions as the wild type strain. Furthermore, essentially all RNA polymerase activity of the protoplasts could be recovered from the heavy fraction after sonication in 0.4 M (NH,),SO, according to Adman et al. (19). We have, therefore, tried to prepare nuclei
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according to the methods described by Tonino and Rozijn (36), or by Bhargava and Halvorson (37). Although the first method yielded relatively clean nuclei the difference in activity between nuclei isolated from protoplasts incubated with casamino acids and starved protoplasts was always relatively small and never exceeded a factor two. Later we found that polyvinyl pyrrolidone employed in this method inhibits endogenous RNA polymerase activity of yeast nuclei very strongly, an observation also made by Halvorson and colleagues (32). The method of Bhargava yielded in our hands only very small and variable amounts of nuclei of very low activity. Therefore, we have tried to develop a method for the preparation of relatively clean nuclei which still show a substantial difference in activity for RNA synthesis depending on the original incubation medium for the protoplasts. The method is essentially an adaptation of the procedure described by Lee and Byfield (38), for the isolation of macronuclei of Tetrahymena pyriformis. Based on the DNA content, we have obtained ninety per
FIG. 1. Induction of RNA synthesizing activity of a yeast 10,OOOg sediment after the addition of casamino acids to a protoplast suspension starved for amino acids. Yeast protoplasts (final concentration 1 mg of protein/ml) were incubated for 60 min at 30°C in 30 ml of medium without casamino acids. Casamino acids were added at zero time (final concentration 0.2%) and 4-ml samples were withdrawn at the times indicated and immediately cooled in ice. A lO.OOOg sediment was prepared and incubated with 3H-labeled UTP as described in the text (a). For the determination of the RNA synthesizing capacity of the whole protoplasts (b), 0.5-ml samples were withdrawn and incubated with 10 fig of ‘H-labeled adenine (sp act 0.1 &i per rg) for 5 min at 20°C. The incubation was terminated by the addition of 5 ml of cold 5% trichloroacetic acid. Radioactivity in RNA was estimated as described before (25).
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cent recovery of the nuclei. The nuclei were judged relatively free of attached cytoplasmic material, although we have never been able to remove all adhering cytoplasmic contaminants. The density of the nuclei was found to be very dependent on the nature of the medium in which the protoplasts had been previously incubated. Upon centrifugation through a stepwise sorbitol gradient, nuclei from starved protoplasts were found to band at about 60% sorbitol, whereas nuclei from protoplasts which had been incubated with casamino acids were much denser and banded between 70 and 80% sorbitol. This indicates that complete separation of the nuclei from other cell constituents which were not or were less influenced by the medium by centrifugation through stepwise sorbitol gradients is not easily attained. The nuclei were found to contain about four per cent of the total RNA of the cell. An analysis of the nuclei prepared by the present method gave an average DNA, RNA, protein ratio of about 1:3:30, in reasonable agreement with the values reported by Tonino et al. and Bhargava et al. (36, 37). These values were again dependent on the medium in which the protoplasts were incubated. Although the protein to DNA ratio of the nuclei did not change substantially, the ratio between DNA-and RNA was-found to fluctuate between 1:4 for nuclei incubated in rich medium against 1:2 nuclei from starved protoplasts. This difference may well be the reason for the difference in density described above. Nuclei isolated by the present method readily incorporate labeled nucleoside triphosphates into RNA. Moreover, such nuclei showed, like the 10,OOOg sediment mentioned before, considerable difference in activity depending on whether they had been prepared from protoplasts which had been starved or incubated with amino acids. In the present paper, we will describe some of the properties of such yeast nuclei, and the RNA which is synthesized. At 3O”C, yeast nuclei synthesize RNA at a rapidly decreasing rate (Fig. 2). The system virtually WaSeS t0 inCOrpOI%tC? labelled UTP after 10 minutes. At 20°C the
AND
BELTZ
system retains its activity considerably longer. The total amount of radioactivity which is incorporated into RNA at 20°C was found to exceed the amount incorporated at 30°C but more important, the RNA synthesized at the lower temperature was found to consist of high molecular weight material sedimenting between 5 and 35s whereas, the RNA synthesized at 30°C consists mainly of low molecular weight material (Fig. 3). Apparently, the RNA synthesized at the lower temperature is also rather stable, for no change in sedimentation profile could be observed when the incubation time was extended from 5-15 min. In attempts to characterize the system further, we have determined the effects of a number of cofactors and inhibitors. The system was found to be dependent on magnesium ions, although the optimum concentration was found to have a rather broad range, between 0.005 M and 0.02 M. Manganese which is indispensable for optimum activity of the isolated eucaryotic DNA dependent RNA polymerases (17, 19) was found to inhibit strongly. Salts of monovalent cations were found to stimulate slightly at low concentrations, at
10
20
30
Tlrnt-~rnllll FIG.
2. Time
course of yeast nuclear RNA syntheat 20” and 30°C. Yeast nuclei were isolated and incubated with nucleoside triphosphates as described under Methods at 20°C (W-----B) and 30°C (04). The total volume of the incubation mixture was 1 ml. Samples (0.1 ml) were withdrawn at the times indicated and the radioactivity incorporated into RNA was estimated as described in the
sizing activity .
text.
RNA
SYNTHESIS
IN YEAST
327
NUCLEI
FIG. 3. Sedimentation pattern of RNA synthesized by isolated yeast nuclei at 20” and 30°C. Nuclei were isolated from protoplasts which had been preincubated with 0.2% casamino acids for 60 min at 30°C. The isolated nuclei were incubated with nucleoside triphosphates for five minutes at 30°C (a) or 20°C (b) as described under Methods. RNA was isolated with phenol SDS and sedimented through sucrose in DMSO.
higher concentrations, (over 0.2 M) the activity of the isolated nuclei was inhibited. Moreover, at increasing concentrations of ammonium sulphate, the difference in activity of nuclei isolated from protoplasts, which were starved or incubated with amino acids, became less and less. The optimal pH of the system was found to be about 7.5. Actinomycin D was found to inhibit very strongly, but (Yamanitin was found to have only marginal effects. These results are summarized in Table I. Based on these results we have taken as optimal conditions for incubation 5 min at 2O”C, 0.006 M MgCl, and no manganese. The RNA synthesized by the nuclei at 20°C was analyzed further for the presence of low molecular weight RNA and polyadenylic acid containing messenger RNA. Figure 4 shows that after prolonged centrifugation of the RNA in sucrose gradients in DMSO, no detectable newly synthesized material sediments in the region of the low molecular weight RNA components indicating that the system is deficient in the formation of low molecular weight RNA species, such as transfer RNA. For the estimation of the capacity of the system to synthesize poly A containing messenger R:NA, we have made use of the fact that such RNA binds to certain types of unmodified cellulose (32, 33). Figure 5 shows that, indeed, a fraction of the RNA (averaging about 15 per cent) binds to cellulose in 0.5 M KCl. Subsequent investigation of the sedimentation properties of
TABLE
I
THE EFFECTS OF SOME MONO- AND DIVALENT CATIONS AND SOME INHIBITORS ON RNA FORMATION BY ISOLATED YEAST NUCLEI” Complete
system
cpm/lOO ccg nuclear protein 3400 5600 7400 7500 7700
+ + + + +
5 x 1O-3 5 x lo-* 2 x
+ + + +
5 x lo-’ M MgCl, + 10m3 M MnCl, 5 x lo-’ M MgCl, + 5 x lOma M MnCI, lo-* M MgCl, + 1Om3 M MnCl, lo-* M MgCl, + 5 x lo-’ M MnCl,
740 520 1530 1140
+ + + +
10m2 M MgCl, lo-’ M MgCl, lo-’ M MgCl, lo-* M MgCl,
8300 8600 9700 6300
+ lo-* + lo-’
lo-’
M MgCl, MgCl, lo-’ MM&l2 M MgCl, lo-* MMgCl, M
M MgCl, M MgCl,
+ + $+
0.05 M KC1 0.1 M KC1 0.2 M KC1 0.4 M KC1
+ actinomycin D (2 Ng) + amanitin (4 rg)
1300 7400
a Yeast nuclei (100 pg of protein) were incubated in 0.3 ml of a solution containing 20 pg of unlabeled ATP, GTP, and CTP, 6 pmoles of Tris-HCI buffer pH 7.5, 0.6 pmoles of mercaptoethanol, 1 PCi of JHlabeled UTP, and other additions as mentioned in the table. Samples were incubated for 5 min at 2O”C, and the cold trichoroacetic acid insoluble radioactivity was estimated.
this RNA fraction showed that it sediments at about 14S, close to the value found earlier for yeast messenger RNA synthesized in uiuo (49) (Fig. 6). A further study
328
de KLOET
AND BELT2
In order to study the initiation of RNA synthesis, we have investigated the incorporation of -r32P-labeled adenosine and guanosine triphosphate. The latter nucleotides were the predominant initiating nucleotides in RNA formation by isolated yeast DNA dependent RNA polymerases and we have assumed that the same is the case in isolated nuclei or in uiuo although no proof exists (40). The results of these experiments showed that most of the label Fract,on number that was incorporated into material which FIG. 4. Sedimentation pattern of RNA synthesized by isolated yeast nuclei. Yeast nuclei were remains in the water phase after shaking incubated as described under Fig. 3 and the isolated with phenol is largely insensitive to RNARNA was centrifuged through a sucrose gradient in ase, indicating that it represents a contamDMSO containing 0.01 M KC1 for 30 h at 30,000 rpm. inant. To characterize the incorporated radioactivity further, we have hydrolyzed the RNA in alkali and subjected the hydrolysate to paper chromatography. No label was found to chromatograph in the region where nucleoside tetraphosphates are expected to appear, using nucleoside triphosphates as markers upon chromatography in 75 per cent alcohol according to Lane (41) or in 0.1 M phosphate according to Cashel (42). Finally upon sedimentation through a sucrose gradient, virtually all the labeled material remained in the upper half of the gradient whereas the newly synthesized mainly between 10 and FIG. 5. Fractionation of RNA synthesized by iso- RNA sediments 35s (Fig. 3). These observations indicate to lated yeast nuclei on a cellulose column. Yeast nuclei were incubated with ‘H-labeled UTP at 20°C as us that the mechanism for initiation of described before. The RNA was isolated and fractionRNA synthesis in yeast nuclei is severely
i I 05/
ated by chromatography Feigelson (32).
on cellulose as described by
I
0.8 c -1 80 of the effect of cy-amanitin showed that the amount of polyadenylic acid containing messenger RNA was not affected, even not at concentrations as high as 25 &ml. The data described above show that isolated yeast nuclei can elongate RNA molecules of considerable molecular weight and can complete messenger RNA molecules since polyadenylic acid, which causes messenger RNA to adhere to cellulose, is added posttranscriptionally (39). FIG. 6. Sedimentation pattern of yeast messenger We were therefore interested in whether RNA synthesized by isolated nuclei and fractionated yeast nuclei prepared by the present on cellulose RNA synthesized by isolated yeast nuclei method could carry out other important was fractionated as described under Fig. 5. The steps in RNA formation, like initiation and material eluting with buffer containing no KC1 was processing, e.g., of ribosomal precursor collected and centrifuged through a dimethylsulfoxide RNA. sucrose gradient.
RNA
SYNTHESIS
impaired, although we cannot exclude that in yeast nuclei terminal phosphates of the initiating nucleotides have a very short half life which makes it impossible to detect such phosphate residues by the present techniques. The conclusion that isolated nuclei cannot initiate RNA synthesis is in agreement with the conclusion reached by other investigators for other eucaryotic nuclei (43, 44). Processing of ribosomal RNA precursors was studied by pulse labeling in uiuo followed by a continued incubation of the labeled nuclei. in uitro. The data in Fig. 7 show that no detectable processing of ribosomal precursor RNA occurs under the present experimental conditions, both when 8-3H-labeled adenosine or [Me3H]methionine are used as labeled precursors. The continuous drop in RNA synthesizing capacity of the nuclei during incubation at 20°C or 30°C might, therefore, well reflect the inability of the nuclei to initiate RNA sythesis coupled to a continued elongation and termination of the formation of RNA molecules. Indeed, as Table II shows, the rate at which the nuclei loose their capacity of RNA synthesis is reduced at a higher rate when incubated in the presence of nucleoside triphosphates than when incubated in the absence. This might be
t0
20
10 Froctlon
20
number
FIG. 7. Stability of preformed RNA in nuclei upon reincubation in vitro. Yeast protoplasts were preincubated in casamino acids containing medium for 60 min at 30°C and subsequently labeled for 2 min with 8-sH-labeled adenine. Nuclei were isolated and reincubated for 10 min at 20°C with unlabeled triphosphates as described in the text, replacing the labeled UTP by 20 pg of the unlabeled compound. RNA was isolated and centrifuged through sucrose DMSO as described. (a) Pattern before reincubation; (b) pattern after reincubation.
IN YEAST
329
NUCLEI
taken as a further indication that lack of capacity to initiate RNA synthesis is the cause of this reduction of the activity of the nuclei. A further analysis showed however that such an incubation of nuclei with nucleoside triphosphates also reduces the activity of the isolated RNA polymerases very strongly, demonstrating that lack of capacity to initiate RNA synthesis as well as inactivation of RNA polymerase causes the drop in activity of the nuclei (Table II). The RNA synthesizing activity of the nuclei, like the 10,OOOg sediment, increases very rapidly when protoplasts which had been starved for amino acids are reincubated in a medium containing amino acids. The increase is completely inhibited by cycloheximide. In this respect, the increase in activity resembles the formation of an induced enzyme. The increase in activity of RNA synthesizing capacity of the nuclei might be due to an increase in activity of the RNA polymerizing enzymes TABLE Loss AFTER
OF RNA INCUBA~ON
II
SYNTHESIZING CAPACITY IN NUCLEI WITH NUCLEOSIDE TRIPHOSPHATES’
cpm/lOO Pg of nuclear protein (a) (b) (c) (d) (e) (f)
Nuclei not preincubated Nuclei preincubated for 10 min Nuclei preincubated without NTP’s Isolated RNA polymerase from (a) Isolated RNA polymerase from (b) Isolated RNA polymerase from (c)
940 430 2370 370 1550
“Yeast nuclei (100 rg of protein) were preincubated at 20°C in 0.25 ml of a solution containing 20 pg of ATP, GTP, and CTP and 1 pg of unlabeled UTP, 6 pmoles of Tris-HCl buffer pH 7.9, 0.6 pmoles of mercaptoethanol, 3 pmoles of MgCl, under conditions as indicated in the table. For the estimation of the activity of the nuclei 0.05 ml of a solution containing 20 pg of unlabeled ATP, GTP, and CTP and 1 bg of “H-labeled UTP (sp act 3 &i/pg) was added after 10 min of preincubation and the incubation continued for another 10 min. For the isolation of the RNA polymerases the amounts of material in the experiments were scaled up tenfold and the enzymes isolated by sonication and precipitation with ammonium sulphate as described under Methods. The activity of the enzyme was tested after dialysis as described in the text.
330
de KLOET
as well as to an increase of the actual amount of the enzymes. Figure 8 shows that two DNA dependent RNA polymerases can be readily extracted from nuclei prepared with Triton X 100. The data on the effect of cy-amanitin show that the elution pattern upon chromatography on DEAE sephadex is similar to the pattern observed by others (19-21) with two components (AI and AII) which are not sensitive to the antibiotic, and a major components B which is sensitive to cy-amanitin. A fourth component eluting after B, also a-amanitin insensitive was infrequently observed, which might be just the result of the concentration dependence of the activity of this enzyme (45). Table III shows that when the RNA polymerases are isolated, the total activity of the enzymes, as well as the ratio between cu-amanitin resistant and sensitive enzymes remains unchanged demonstrating that only the activity of the RNA polymerases is affected as a result of the change in incubation conditions, a conclusion similar to that reached by Gross and Pogo (28) in their studies on RNA synthesis by nuclei of S. cerevisiae.
FIG. 8. Elution pattern of RNA polymerase isolated from yeast upon chromatography on DEAE Sephadex. RNA polymerizing enzymes were isolated and fractionated as described under Methods. The activity of the fractions of the eluate was estimated by incubating 0.05-ml samples for 30 min at 30°C with 0.2 ml of a solution containing 20 rg of unlabeled ATP, CTP, and GTP, 5 rmoles of Tris-HCl buffer pH 7.9, 0.5 nmoles of MnCl,, 1.5 rmoles of MgCI,, 4 rmoles of mercaptoethanol, 20 pg of denatured calf thymus DNA and 0.5 &i of Ha-labeled UTP. The reaction was stopped by the addition of 5 ml cold 5% trichloroacetic acid.
AND
BELTZ TABLE
III
ACTNITY OF ISOLATED RNA POLYMERASES ISOLATED FROM YEAST NUCLEI PREPARED FROM PROTOPLA~TS WHICH HAD BEEN STARVED OR INCUBATED WITH AMINO ACIDS” CPm/loo CcfT of nuclear protein Nuclei Nuclei with
from starved protoplasts from protoplasts incubated casamino acids
RNA polymerases isolated starved protoplasts +a-amanitin (2rg)
from
RNA polymerases from protoplasts incubated with amino acids +cu-amanitin (2 ng)
1470 11700
8317 5637 8737 5314
’ Yeast nuclei were isolated from protoplasts which had been either starved for 60 min by incubation in the absence of casamino acids or which had been incubated in basic medium with the addition of 0.2% casamino acids. The RNA synthesizing capacity of the nuclei was tested as described under Methods. The isolation of the RNA polymerase activity and the estimation of the enzymatic activity has been described in the text.
A change in activity of the RNA polymerizing activity of the nucleus may be the result of a change in the rate at which individual enzyme molecules catalyze the formation of RNA molecules. It may also be that under conditions of amino acid starvation part of the polymerase is unavailable for transcription of endogenous DNA, and possibly available for the transcription of added foreign DNA. The latter possibility was investigated. As was shown above manganese ions inhibit the endogenous RNA synthesizing activity of the nuclei, although the isolated RNA polymerases depend heavily on the presence of this ion for optimal activity (Ii’, 19). Table IV shows that the nuclei from starved protoplasts show a more substantial increase in activity upon the addition of denatured calf thymus DNA compared to nuclei isolated from protoplasts incubated in a rich medium. The latter result suggest that activation of latent RNA polymerase is at least in part, responsible for
RNA SYNTHESIS TABLE
IV
THE EFFECT OF THE ADDITION OF DNA ON RNA SYNTHESIS BY ISOLATED YEAST NUCLEP
cpm/lOO I.%of nuclear protein
Nuclei from protoplasts incubated without casamino acids + 20 pg of denatured calf thymus DNA Nuclei from protoplasts incubated with casamino acids + 2Opg of denatured calf thymus DNA
990 2770 5600 5100
“Yeast protoplasts (1 mg of protein/ml) were preincubated at 30” in lo-ml medium without casamino acids. After 60 min casamino acids (final concentration 0.2%) were added and the incubation continued for another 30 min. Nuclei were isolated and incubated for 5 min at 20°C in 0.3 ml of a medium containing 20 rg of unlabeled ATP, GTP, and CTP, 6 rmoles of Tris-HCl buffer pH 7.9, 0.6 rmoles of mercaptoethanol, 3 pmoles of MgCl,, 0.6 rmoles of MnCl,, 1 PCi of [3H]UTP and denatured calf thymus DNA as indicated.
the increase in RNA synthesis upon the addition of amino acids to the medium. In yeast, like in other microorganisms, not only the rate of RNA formation is subject to environmental control, but also the ratio in which ribosomal, messenger and transfer RNA are formed. Messenger RNA is in general relatively less influenced than ribosomal RNA formation. A similar result is obtained when the composition of the RNA synthesized by the isolated nuclei
Frocfvsn
IN YEAST NUCLEI
331
is studied. Upon analysis by cellulose chromatography, it was found that RNA synthesized in nuclei isolated from yeast protoplasts which had been incubated in the presence of amino acids contains relatively less polyadenylic acid containing messenger RNA than RNA synthesized by nuclei isolated from starved protoplasts. (15 vs 30%). The data in Fig. 9 show that the former RNA contains more material sedimenting ahead of ribosomal RNA in a dimethylsulfoxide sucrose gradient, which is the region of the ribosomal precursor RNA. Therefore, although the nuclei as isolated by the present procedure are certainly deficient in a number of aspects of RNA synthesis, the composition of the synthesized RNA reflects still the activity of the original protoplasts for the preparation of the nuclei. DISCUSSION
The present study shows that isolated yeast nuclei continue to synthesize RNA for a short period. The observation that more RNA is made and that it has a higher molecular weight when the nuclei are incubated at a low temperature (20°C) than when it is synthesized at a higher temperature (30°C) resembles the observations made by Marzluff et al. (14) in their studies on RNA synthesis by mouse myeloma nuclei. Again, like has been found previously by Tata and others (43), manganese ions inhibit nuclear RNA synthesis although this
number
FIG. 9. Sedimentation pattern of RNA synthesized by yeast nuclei. Yeast nuclei were isolated from protoplasts incubated with or without amino acids and incubated for 5 min at 20°C as described under Methods. RNA was isolated and centrifuged through sucrose in DMSO as described. (a) Sedimentation pattern of RNA synthesized by nuclei isolated from protoplasts starved for amino acids. (b) Sedimentation pattern of RNA synthesized by nuclei isolated from protoplasts incubated with amino acids.
332
de KLOET
divalent cation is necessary for the activity of the isolated enzymes (17, 19-21). Contrary to what has been found for nuclei of other organisms, we have never observed any substantial effect of a-amanitin on RNA synthesis in yeast nuclei, although actinomycin D inhibits very strongly. Although it cannot be ruled out that (Yamanitin cannot penetrate yeast nuclei under the present conditions, another explanation might be that in yeast nuclei messenger RNA as well as the other RNA species are made by a-amanitin resistant enzymes. A somewhat similar but not so extreme conclusion has been reached by Zylber and Penman (15) who concluded that heterogeneous nuclear RNA in HeLa cells is synthesized by two distinct enzymes, one sensitive, one insensitive to the antibiotic. These results are somewhat different from those obtained by Gross and Pogo (28) in their studies on nuclei of S. cereuisiae. These authors observed a substantial effect of cr-amanitin. In their experiments mangenese was included in the incubation medium and the incubation time was substantially longer (30 min) than in the present study. In our studies we have found however that manganese inhibits the activity of the nuclei and consequently we have omitted it from our incubation medium, in order to maintain optimum conditions for RNA formation. Compared to nuclei isolated by others from different organisms (13, 45, 46), yeast nuclei show differences and similarities with respect to a number of important aspects of the biosynthesis of RNA. Based on our experiments with y-labeled [“‘PInucleoside triphosphates yeast nuclei do not seem to initiate the synthesis of new RNA molecules, although the possibility that the terminal phosphate is cleaved off shortly after initiation makes this conclusion at least tentative. The conclusion is however in agreement with that reached by others in their studies of RNA synthesis in isolated mammalian nuclei (45, 44). The decrease in RNA synthesis with time cannot only be ascribed to a failure of the mechanism of initiation, since we have also observed that the activity of the isolated RNA synthesizing enzymes drops
AND
BELTZ
sharply during incubation of the nuclei with nucleoside triphosphates. Earlier studies (8, 9, 48) had shown that in uiuo the ribosomal precursor in yeast sediments as material of about 38S, whereas messenger RNA sediments with an average sedimentation coefficient of 14s (49). Based on the sedimentation data and the chromatographic properties on cellulose, both ribosomal precursor RNA and messenger RNA are synthesized by isolated yeast nuclei. No low molecular weight RNA has been found among the products. Whether this means that transfer RNA is synthesized in yeast as part of a large precursor which is not processed in the course of the experiment or whether the system is actually deficient in the transcription of this RNA component cannot be concluded from the present data. No processing of ribosomal precursor RNA has been shown to occur in isolated yeast nuclei, although isolated nuclei from amplibia (13, 50-52) and certain insects (53) can carry out a number of steps required for the maturation of ribosomal RNA. A major factor in the regulation of the formation of ribosomal RNA in eucaryotes operates through control of the maturation process, which is closely connected to the ability of the cell to carry out protein synthesis. Most likely this control is indirect and related to the availability of certain proteins. In yeast nuclei such a pool might be smaller than in other organisms. On the other hand, the in vitro processing of ribosomal precursor RNA by isolated nucleolar endonuclease (48) has been shown to be strongly dependent on the concentration of divalent cations, so that our incubation conditions might be far from ideal to observe any processing. The finding that labeled messenger RNA can be isolated by cellulose chromatography shows that the nuclei can complete the synthesis of at least this RNA species, since the polyadenylic acid moiety is added post transcriptionally (39). The present study shows that the higher rate of RNA synthesis in the nuclei isolated from protoplasm incubated in the presence of amino acids as compared to the rate in nuclei isolated from starved protoplasts
RNA
SYNTHESIS
can at least in part be explained as being the result of the binding of more RNA polymerase in a form which makes it unavailable for the transcription of added template, and which allows it to transcribe only endogenous DNA. Whether this is accompanied by any additional structural change or change of protein content and composition of the nuclei is at present under investigation. ACKNOWLEDGMENTS This work was supported by American Cancer Society and National Science Foundation. debted to Dr. T. Wieland for a-amanitin.
Grants NP 80 of the NB Pi B 2200 of the The authors are inthe generous gift of
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