Regulation of RNA synthesis in Neurospora crassa

Experimental

Cell Research 99 ( 1976) 245-252

REGULATION

OF RNA SYNTHESIS

NEUROSPORA An Analysis E. STURANI, M. Cl. COSTANTINI,

IN

CRASSA of a Shift-up

R. ZIPPEL, and F. A. M. ALBERGHINA

Centro di Studio de1 CNR per la Biologia Cellulare e Molecolare de& Piante. Istituto di Scienze Botaniche, Universitri di Milano. 20133 Milano, Italy

SUMMARY A shift-up transition of growth from acetate to glucose is analyzed in Neurospora crassa. The rates of DNA and of protein accumulations remain at the preshift values for about 2 h, afterwards they increase to the rate characteristic of the new medium. The rate of RNA accumulation increases markedly 30 min after glucose addition initially at a rate greater than that of the new exponential growth which is achieved later on. An increase of the level of ribosomal proteins accompanies the increase of the rRNA content of the shifting cells, and 2-2.5 h after the shift the ribosomal level has reached the value characteristic of the new steady state of growth. The rate of rRNA methylation, which is strictly proportional to rRNA synthesis, remains almost unchanged in the 30 min following the shift; thereafter it increases to values greater than the final rate. It is interesting that the rate of rRNA synthesis is enhanced above the value typical of the new steady state as long as the ribosome level in the cells is below that characteristic of the new steady state, as if a compensatory mechanism were active.

In Neurospora the rate of synthesis of stable RNA is regulated by environmental conditions. At any given temperature during exponential growth the nutrients present in the medium determine the growth rate of the culture and a proportional enrichment of the cellular level of RNA is observed by increasing the growth rate [l]. This pattern of response is very similar to that described for enteric bacteria by Maaloe [2], but the molecular mechanisms by which it is achieved are likely to be different due to the presence of the nucleus and of different types of RNA polymerase in eukaryotic Neurospora cells [3]. That the regulation of ribosomal RNA synthesis in Neurospora, although qualita-

tively similar to that found in bacteria, is achieved by using different control devices is shown by a study of a shift-down transition of growth during which the synthesis of rRNA is severely inhibited [4] while no guanosine 3’-diphosphate, S’-diphosphate (ppGpp) is formed [5]. The involvement of ppGpp in the negative control of rRNA synthesis in E. cofi is supported by in vivo [6-8] as well by in vitro studies [9-l 11. Other differences have to be expected in shift-up transitions of growth. In fact the increase of the rate of rRNA synthesis observed in bacteria following a shift-up [ 121 is largely due to a shift of RNA polymerase molecules from mRNA genes to ribosomal cystrons [13, 141. This mechanism, of Ex[> Cdl

Rr\

99 (1976)

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course, is not compatible with the known molecular differences and specificities of product

formation

of Ihe RNA

polymerases

of eukaryotic cells [ 151. So if Neuvospora cells are able to adjust their rate of stable RNA synthesis to that of a richer nutritional condition, we expect to find a somehow different mechanism. We already have evidence that the addition of a richer nutrient to Neurospora cultures induces a transition which ultimately results in a more rapid steady-state of exponential growth [16]. In the present paper we report the results of experiments in which we determine the kinetics of growth and of macromolecular accumulations and, more specifically, the rate of ribosomal RNA synthesis during a shift-up transition of growth in Neurospora. The addition of a nutritional supplement is followed, 30 min later, by a preferential stimulation of the rate of synthesis of rRNA, while the rates of protein and DNA synthesis increase over the preshift values much later. The length of the lag period for the stimulation of rRNA synthesis seems too long to be accounted for only by the time required for the synthesis of new RNA polymerase molecules. MATERIALS

AND METHODS

Growth conditions Mycelia of wild type Neurospora crassa 74 A were grown in mineral Vogel medium [17] with the addition of one of the following carbon sources: 2% glucase, 40 mM sodium acetate, 2% glycerol or 2% ethanol. 750 ml flasks containing 200 ml of medium, or 300 ml flasks containing 80 ml of medium, were inoculated with Neurospora conidia (about 2 X lo53 X I05/ml) so that the initial AJhOwas 0.02-0.06. In the case of growth on glucose, acetate or glycerol, the flasks were incubated for I5 h in a Dubnoff water bath at 16°C with shaking at about 100 rpm [16]. Then the temperature of the bath was raised to 30°C and the growth allowed to continue at this temperature. In the case of the growth on ethanol the low temperature incubation was omitted and the flasks were incubated immediately at 30°C. Exp Cell Res 99 (1976)

To induce the shift-up the culture growing in acetate (Ads,, about 0.l50-0.250) was made 2% glucose by

adding 0.05 vol of 40 % sterilized glucose. The growth was monitored as increase in A,,, and the constant of the rate of growth, K(h-i) was determined as prevtously reported [l6]. The number of duplications per hour (CL)was calculated from: K/ln2.

Chemical determination

of RNA and DNA

RNA and DNA contents of the culture have been determined as previously described [I] by the orcinol and diphenylamine methods [18, 191.

Incorporation of radioactive precursors into nucleic acids and protein To measure the incorporation of [3*P]orthophosphate mto nucleic acids the cultures were grown in a modified Vogel’s medium containing 2 mM [32P]orthophosphate (spec. act. 0.05 Ci/mole). It has been shown that this concentration of phosphate does not modify the growth rate nor limit growth in our experimental conditions [ 161. The radioactivity incorporated into cold trichloracetic (TCA)-precipitable material subtracted by that precipitable in hot TCA, i.e. essentially into RNA, was measured as previously described [4]. To determine the protein net synthesis the cultures were grown in the presence of 5x lO-4 M [carboxyY]L-leucine (0. I Cilmole) and of 5 X 10e4 M L-isoleucine, which was added to overcome a certain inhibition of the growth rate observed in acetate in the presence of leucine. The radioactivity incorporated into hot TCA-precipitable material was determined as previously described [4]. To determine RNA methylation [methyl-i4C]Lmethionine (spec. act. I I.5 Cilmole, obtained from New England Nuclear Corporation) was added to the culture (200 ml, A 450=0.3) to a final concentration of 5 X IO-’ M. Under our experimental condition this concentration was not rate-limiting for methionine incorporation into purified RNA, although it was ratelimiting for methionine incorporation into the cellular soluble fraction, as one has to expect due to the fact that the Km for methionine uptake in Neurospora is 2X 10m5M [20]. We have in fact observed that by increasing the L-methionine concentration, the rate of methionine uptake into the cell increased, while both the rate of radioactivity incorporation into TCAprecipitable material and the rate of incorporation of radioactive methyl into RNA remained unchanged. Besides, the methyl incorporation into RNA was a linear function of time for at least 0.25 duplication times. For the experiments shown in fig. 3 the labeling time was 0.08 duplication times, that is 10 min in glucose, 12 min in acetate, 19 min in glycerol and 37 min in ethanol. For the shift-up experiments 80 ml cultures were labeled for 5 min with 5 x IO-” M [methyl3H]L-methionine (SOCilmole). The cultures were then rapidly collected by filtration, washed with cold water, and quickly used for RNA extraction.

Analysis of a shift-up in Neurosportr

I 2

1

methyl incorporated during I min into the rRN.4 fraction corresponding to I A,,, unit of total RNA, was multiplied by the RNA to DNA (w/w) ratio characteristic of Neurosporu cells in exponential growth on each medium [I] so as to obtain the pmoles of methyl incorporated, min’/l A,,, unit of DNA (45 pg of DNA) [2l]. From this value, the molecular weight of a haploid genome of Nrurosporcl crust, being 1.3~ 10’” [22], the number of methyl groups incorporated. min’/genome was calculated.

Ribosomal

-2

-1

0

1

2

3

4

Fig. 1. Abscissu: time (hours); ordinafe: (left) A,,,, (a, A); (right) 32Pincorp. into cold minus hot (0, 0) and [l-‘4C]leucine incorp. (0, n ) into hot TCAprecipitable material. A, 0, Cl, Control in acetate: A, 0, n , after adding glucose. Growth, nucleic acids and protein accumulation during an acetate to glucose shift-up. Cultures exponentially growing in acetate were shifted-up by addition of 2% glucose. Growth as A,,,, [a*P]orthophosphate and [ I-14C]leucine incorporations were monitored. [32P]orthophosphate and [“C]leucine were added at the moment of inoculation.

RNA purzjkation

content

In order to determine the ribosomal content of the mycelia during the shift-up transition, 200 ml cultures were grown in the conditions used for the continuous labeling of proteins (see the previous section): 0.5 mM [carboxy-‘4C]L-leucine, 0.05 Cilmole, and 0.5 mM I.-isoleucine were added to the acetate medium. At the times desired, before and after glucose addition, the cultures were quickly collected by suction. washed with cold water and frozen in liquid nitrogen. Ribosomes were extracted as previously reported [I], with the only difference that the I5 000 g pellet was washed once with the extracting buffer. The combined supernatants after a mild RNase treatment (S @g/ml for I5 min at 4°C) were analyzed on sucrose gradients [I]. The ribosomal content was calculated as the ratio between the hot TCA-precipitable radioactivity of the ribosomal particles and the total radioactivity of the gradient (i.e. of the 15000 g supernatant).

RESULTS Levels of DNA, RNA and protein in Neurospora cultures during (1 shift-up

cultures exponentially growing at 30°C on acetate medium have a growth

Neurospora

andfractionation

RNA was extracted in the cold by the method previously reported employing the pH 7.4 buffer and a phenol-cresol mixture [4]. A known aliquot of the puritied RNA (5-10 A?,,, units) was layered on a I2 ml linear sucrose gradrent (5-20% w/w) prepared in 0.025 M Tris-Cl pH 7.4, 0.1 M NaCl, I pg/ml polyvinylsulfate. The gradients were centrifuged at 1°C at 24000 rpm for 16 h in the SW40 rotor of the Spinco L-2-65B centrifuge, and fractionated on an ISCO density gradient fractionator connected to a UV analyzer. The cold TCA-precipitable radioactivity of the fractions was determined as previously reported. The top fractions of the gradient containing sRNA were deacylated by incubation at pH 8.8 before precipitating with TCA [4].

Calculation of rRNA

947

of the rate of methylation

The rates of methylation (expressed as number of methyl groups incorporated in rRNA, min-‘/genome) were calculated as follows. The number of pmoles of

Fig. 2. Abscissa:

time (hours); ordinate:

(left) DNA

n , 0; (righf) 0, 0, RNA content (&ml of culture). 0, 0, Control on acetate; 0, n , after adding glucose.

RNA and DNA levels during an acetate to glucose shift-up. Conditions as in fig. 1. At the time indicated RNA and DNA content of the cultures were determined by orcinol and diphenylamine methods, respectively. Exp

Cdl

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rate (K, hh’) of 0.28 [ 11. As shown in fig. 1 the growth is balanced as the absorbance at 4.50 nm (A&, the content of nucleic acids (continuously labeled with [32P]orthophosphate) and that of protein (continuously labeled with [carboxy-14C]L-leucine) increase with the same rate constant. At the time indicated as zero in fig. 1 glucose is added to the culture to make its final concentration 2 % (w/v). The rate of growth, measured as AdsO,is unaffected by the change in environment for about 2 h, then slowly increases to the value characteristic of cells growing in glucose (K= 0.35). Protein accumulation follows the pattern indicated for A,,,: only after about 2 h the rate of protein net synthesis increases above that of the cells growing in acetate (fig. 1). The incorporation of 32P into the cold TCA-precipitable material, subtracted by the hot TCA-precipitable material, measures in Neurospora RNA accumulation, as in our conditions DNA represents only about 3 % of the total nucleic acids [l]. Fig. 1 shows that the rate of RNA accumulation does not change for about 45 min, then it strongly increases. It is interesting to note that the initial rate of RNA net synthesis is substantially greater (K= 0.56) than that typical for cells growing in glucose (K=O.35), which is measured several hours after the shift-up. The chemical determinations of RNA and DNA reported in fig. 2 confirm, for RNA, the findings of fig. 1 and, for DNA, indicate a pattern very similar to those observed for the protein and for AdsO. In conclusion. during a shift-up a dissociation of macromolecular syntheses occurs: DNA and protein syntheses proceed at the preshift rates for a little more than 2 h, while the synthesis of stable RNA is early (just 45 min after the transition) and specifically stimulated. Exp CellRes 99 (1976)

In the experiments recorded in the following sections we measure the rate of synthesis of mature methylated rRNA during the shift-up and show that the stimulation of the total RNA accumulation previously observed (figs 1 and 2) is due chiefly to a strong enhancement of the rate of rRNA synthesis. Moreover, we show that there is an increase in the content of ribosomal proteins parallel to that of rRNA. Rate of rRNA methylation at different rates of growth The measurement of the methylation rate of rRNA allows a correct determination of the rate of rRNA synthesis as shown by the following experiment. Cultures of Neurosporu growing exponentially in different media are labeled with [methylJ4C]L-methionine under conditions which ensure, as indicated in the Methods, that the amount of radioactivity incorporated into phenol-extracted, sucrose-density gradient fractionated rRNA gives a correct measurement of its rate of methylation. The rates of rRNA methylation (expressed as methyl groups incorporated into rRNA, min-‘/genome) calculated as described in the Methods from the radioactivity incorporated into rRNA, are reported in Iig. 3 (closed circles). In the same figure are given the rates of synthesis of rRNA (open circles), expressed as nucleotides polymerized, min-‘/genome for eight conditions of exponential growth taken from a previous paper [l]. In the same paper it has been shown that the rate of rRNA synthesis (rr) is the function of the growth rate (p) according to the following equation: r,=6.51 x 107$1g which of course yields a straight line on a

Analysis of a .yh(ft-up in Neurosportr -5

-1

-05

I

I 0.5

1

Fig. 3. Abscissa: rate of growth (p); ordinate: (left) nucleotides (0) min-‘/genome (x IO-‘); (righf) methyl (0) min-r/genome (X 10ms). Rate of synthesis and of methylation of rRNA as a function of the rate of growth. Mycelia were grown in ethanol (p=O. 13); glycerol (~=0.26); glycerol+ casamino acids (~30.48); acetate (~=0.41); acetate+ casamino acids (~=O.SS); glucose (r=OSl), glucose+ casamino acids (~=0.65); nutrient broth (~=0.91). The rates of synthesis of rRNA (0) are expressed as nucleotides polymerized, min’lgenome [I]. The rates of methyl incorporation into rRNA (O), expressed as methyls incorporated, min’lgenome, were determined as indicated in Experimental Procedures. The data are the average of three independent determinations whose values did not differ more than f5 %.

249

glucose were shortly (5 min) labeled with [methyl-“H]L-methionine. RNA was phenol extracted, deacylated and the amount of methyl incorporated into 1 Az6,,unit of total RNA was determined (fig. 4A). Then RNA was fractionated on sucrose gradients. Fig. 4B, C reports the methylation rates of both tRNA and rRNA, expressed as methyl incorporated in tRNA and rRNA per A.,,, unit of unfractionated RNA. From the data of fig. 4 it appears that the methylation rates of both stable RNA species are stimulated following the shift-up -that of rRNA much more than that of tRNA.

logarithmic plot as that of fig. 3. The comc I parison of the two measurements indicates that the rate of rRNA methylation is strictly proportional to the rate of rRNA synthesis and therefore offers a reliable way to measure it. The extent of rRNA methylation in Neurospora is not affected by the growth condition and it can be calculated (from Fig. 4. Abscissa: time (hours); ordinate: methyl inthe data of fig. 3) to be about 0.8 methyl carp. (pmoles/A,,, unit of RNA). Rate of methylation of tRNA and rRNA during an acetate to glucose shiftgroup for every 100 nucleotides polymer- up. Cultures growing exponentially in acetate were ized, in agreement with values reported for shifted-up by adding glucose (2 %, final cont.). At different times before and after shift-up the cultures were rRNA of other species [23]. labelled for 5 min with 5~10~~ M [methyL3H]tRate of rRNA methylation during the shift-up Cultures exponentially growing in acetate and at different times after the addition of

methionine. The RNA was phenol-extracted and the radioactivity incorporated into deacylated tRNA and in rRNA was determined. The data are expressed as pmoles of methyl incorporated per Azeo unit of total unfractionated RNA into (A) total RNA; (I?) tRNA; (C) rRNA. Each symbol refers to an independent experiment. Exp Cd R<3 9Y (/ 976 1

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Sturani et al.

reported in fig. 4 and the rates of stable RNA synthesis, calculated (from the data of fig. 1) as increments of the RNA level over short time intervals, are superimposable, the length of the lag and the extent of the overshooting in RNA stimulation being the same in the two determinations. time (hours); ordinate: ribosomal proteins % of S,, proteins. Level of the ribosomal proteins during an acetate to glucose shift-up. Cultures growing exponentially in acetate were shifted by the addition of 2 % glucose. [Carboxyl-14C]L-leucine was added at the momentof inoculation. At each of the times indicated the level of ribosomal proteins was determined as the percentage of radioactivity present in the proteins of the I.5000 g supernatant which is found in ribosomal proteins, fractionated as indicated in the Methods. 0, l , Results of independent experiments. Fig. 5. Abscissa:

The stimulation of the methylation rate of tRNA is greater than the actual increase of its synthesis. In fact we have observed that the extent of methylation of tRNA, always much higher than that of rRNA, varies with growth conditions, and is greater in glucose than in acetate (unpublished results). Moreover, the synthesis rates of tRNA in the two conditions are quite similar [ 11. Instead, the enhancement of the methylation rate of rRNA is quite conspicuous and reflects its synthesis rate, as discussed in the previous section. It is not observed immediately after thk addition of the richer medium, but about 30 min afterwards, and during the first 20 min after the shift-up the rate of rRNA methylation is even slightly lower than the rate in acetate; it reaches a maximum after 100-150 min, then again it declines toward the value characteristic of the growth in glucose. The pattern observed for methylation of rRNA justify the kinetics of RNA accumulation shown in figs 1 and 2: the rates of rRNA methylation measured at the different moments of the growth transition are Exp Cell Res 99 (1976)

Level of ribosomes and of ribosomal proteins during the shift-up

We have previously shown that the addition of glucose induces an enhancement of the rate of rRNA synthesis and accumulation. To investigate whether this increase was accompanied by a parallel increase of the ribosomal proteins to yield ribosomal particles, the experiments reported in fig. 5 were carried out. The mycelia were grown in the presence of [carboxy-14C]L-leucine and during growth in acetate and at different times after the shift-up the radioactivity present in the proteins of the 15000 g supernatant as well as that present in the proteins of the ribosomal fractions were determined. The relative ribosomal protein content, expressed as percentage of the total protein in the 15000 g supernatant, is shown in fig. 5 to increase following glucose addition. These findings indicate that a stimulation of the synthesis of ribosomal proteins takes place parallel to that observed for rRNA to give an enhanced formation of ribosomes. DISCUSSION The data reported in this paper indicate that in Neurospora the transition from an exponential growth in acetate to a faster one in glucose is characterized by early and preferential stimulation of the rRNA synthesis. This appears to be a general behavior, as kinetics essentially similar to that reported in fig. 1 is obtained when 0.2% glucose, instead of 2%, is added to cells in

This synthesis would require a time much shorter than 30 min, as the rate of protein synthesis in Neurosporu cells growing in acetate is I .25x IOH amino acids. min ‘i genome [I]. It seems therefore more probable that this time is required to allow that the changes in the environment may bring about the intracellular metabolic alterations which induce the enhancement of rRNA synthesis. lt is interesting to recall Fig. 6. Ahscissrr: time (hours); or&tare: rate of RNA synthesis. at this point that Carter & Dawes [?S] have Rates of RNA net synthesis per DNA unit during the observed that in yeast cells shifted to a supshift-up. The relative rates of RNA net svnthesis were calculated from the data of fig. 1 as increments of the plemented medium the specific activity of RNA level over I5 min intervals and were referred to RNA polymerase I starts to increase only one unit of DNA (fig. 2). The rate of stable RNA synthesis in acetate is taken as 100. The dotted line indi30 min after the shift. cates the rate of RNA synthesis in glucose [I]. The rates of net synthesis of protein and of DNA start to increase over the preshift exponential growth in acetate, as well as values 2-2.5 h after the shift, i.e. when the when the shift-up is induced by adding sustained RNA accumulation and the accasamino acids to cells growing in glucose. companying synthesis of ribosomal proteins The fairly long lag period (30 min) be- have increased the ribosomal content by a tween the induction of the shift-up and the factor of 1.4 over the acetate level (see fig. actual increase of the rate of rRNA syn- 5). The ribosomal level in acetate being thesis is observed in all transitions we have 2.5~ IO” ribosomes/genome [I]. we calconsidered, from a poor to a rich medium, culate that 2-2.5 h after the shift the riboand it seems therefore to be a typical feature somal level is 3.5 x IO”, i.e. practically the level typical of cells growing in glucose of the shift-up transitions in Neurosporu. De novo synthesis of RNA polymerase I [I]. Thus the balanced exponential growth is likely to take place during the shift-up, as on the new medium starts when, due to the it is known that the amount of RNA poly- dissociation of the macromolecular synmerase I increases with the growth rate theses, the composition typical of the cells [24, 251. However, this lag period is too in the new condition is achieved, in a long to be accounted for only by the time reverse fashion with the pattern observed required for the de novo synthesis of RNA during a shift-down [4]. In such conditions polymerase. It can be calculated that the RNA synthesis stops while the DNA and molecules of RNA polymerase I active in protein accumulations proceed, and it Neurospora cells exponentially growing in resumes with the new rate only when the acetate are about 1500 per genome [I] and macromolecular composition is that of the that about 3 000-4000 more molecules cells in the new slow growth medium. Another interesting point is shown in fig. should support the higher synthesis rate observed after the shift-up. As the molecular 6 (where the relative rate of RNA net synweight of RNA polymerase I is 5X lo5 [26] thesis per DNA unit is plotted during the their synthesis would require the poly- shift): following the nutritional shift-up merization of about 2~ IO7 amino acids. there is an overshooting in the RNA re400’

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sponse, in fact the rate of rRNA synthesis per genome increases to about three times the preshift rate, whereas the rate in glucose is only 50% higher than that in acetate [l]. This enhanced rate is observed as long as the cells have a ribosomal level lower than that characteristic of the new growth condition and the rate begins to decrease when the ribosomal level per genome reaches the value typical of the new growth condition. These and our previous findings on a shift-down [4] strongly supports the idea that a compensatory mechanism is effective in Neurospora which allows to modulate the rate of rRNA synthesis according to the nutrient available to the cells and to the actual level of ribosomes [27]. REFERENCES I. Alberghina, F A M, Sturani. E & Gohlke. J R. J biol &em 250 (1975) 438 I. 2. Maalee, 0. Dev biol. stmol. 3 (1969) 33. 3. Tellez de Ifton, M, Leoni, P ‘D &‘Torres, H N, FEBS lett 39 (1974) 91. 4. Sturani, E, Magnani, F & Alberghina, F A M, Biochim biophys acta 319 (1973) 153. 5. Alberghina, F A M, Schiaffonati, L, Zardi, L & Sturani, E, Biochim biophys acta 3 I2 (1973) 435.

Exp Cell Res 99 (1976)

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