JOURNAL OF BACTERIOLOGY, Mar. 1976, p. 1057-1073 Copyright C) 1976 American Society for Microbiology

Vol. 125, No. 3 Printed in U.S.A.

Escherichia coli Mutants with Altered Ribosomal Ribonucleic Acid Metabolism JAMES M. JACKSON III AND STEPHEN G. CHANEY* Department of Biochemistry, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27514

Received for publication 26 November 1975

This paper reports the partial characterization of two temperature-sensitive mutants of Escherichia coli with alterations in ribosomal ribonucleic acid (rRNA) metabolism at the restrictive temperature. Both mutants continue to synthesize deoxyribonucleic acid and protein at 42 C but showed little or no accumulation of RNA. Both strains are inducible for f3-galactosidase at the restrictive temperature, showing that messenger RNA synthesis continues and that the messenger RNA is translated into functional protein. One of the strains, 2S474, shows a rather severe depression (sevenfold) in the synthesis of all classes of RNA at 42 C. In addition, the synthesis of rRNA is selectively depressed, with the percentage of rRNA made decreasing by 5- to 10-fold (indicating an overall 35- to 70-fold decrease in the rate of rRNA synthesis). This appears to be a specific effect on rRNA synthesis, since the small amount of rRNA which is made at 42 C appears to be processed normally. In the case of the second strain, 2S139, the restrictive temperature has only a minimal effect on total RNA synthesis. However, there appears to be a selective depression in the rate of rRNA synthesis and, possibly, in the conversion ofp16S rRNA to m16S rRNA. The mechanism by which Escherichia coli of these mutants have an altered RNA polymcontrols stable ribonucleic acid (RNA) synthe- erase (3, 16, 19, 26, 31). However, in the cases sis during amino acid starvation has become studied, the synthesis of all classes of RNA fairly well understood in recent years, primar- seems to be affected to the same extent at the ily because of the existence of mutants (the so- restrictive temperature. We have recently recalled "relaxed" mutants) which lack the abil- ported the isolation of a number of temperaity to control stable RNA synthesis during ture-sensitive mutants which, on the basis of amino acid starvation (29). It has been sug- hybridization studies, appear to be specifically gested that stable RNA synthesis may be regu- unable to synthesize ribosomal RNA (rRNA) at lated by levels of MS1 (guanosine 5'-diphos- 42 C (5). In this paper we report the partial phate and 2'- or 3'-diphosphate) (4, 17), which characterization of two of those mutants. is produced in an idling reaction on the riboMATERIALS AND METHODS some in the absence of a required aminoacyl and media. D10 (met-, rnaseI-, relh) has Strains transfer RNA (tRNA) (13, 14). However, the been described by Gesteland (10). 2S139 mechanism by which the cell controls stable and 2S474 werepreviously isolated as temperature-sensitive RNA synthesis during a shift-up or shift-down survivors of a nitrosoguanidine-treated culture of in the growth rates is less well understood. D10 which had been exposed to large doses of Although MS1 levels do rise transiently during [3H]uridine and [3H]guanosine at 42 C (5). They rea shift-down (12, 21) the increase is variable tain all of the other characteristics of the parental (21) and slow compared to the effect of the shift- strain. All strains were carried routinely by resdown on RNA synthesis (9). Obviously, it treaking the strains every 2 to 3 weeks on rich would be beneficial to be able to study mutants medium agar plates. The strains were stored in form in 50% glycerol at -20 C. Both which were altered in the control of stable RNA permanent mutant strains are stable, with reversion frequensynthesis in loci other than the rel locus (which cies less than 10-8. The mutant strains are both controls the response of cells to amino Iacid only ofweakly temperature sensitive. That is, single starvation). colonies will grow on agar plates at 42 C, although A number of mutants have been isolated in much more slowly than at 30 C (in contrast, the recent years which are temperature sensitive parental strain grown more rapidly at 42 C). for RNA synthesis (3, 16, 18, 19, 26, 31). Many The cells were usually grown on M9 medium (1) 1057

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supplemented with 0.2% Casamhio Acids (Difco) and either 0.2% glucose or glycerol. In some experiments they were grown in LB medium (23). D10 grows at 30 C with a doubling time of 55 min on M9 medium plus glycerol, 46 min on M9 plus glucose, and 35 min on LB medium. 2S474 grows with a doubling time approximately 2.2 times as great on all three media, and 2S139 grows with a doubling time 2.6 times as great. The mutant strains are distinctly temperature sensitive on all three media. Materials. [3H]uridine (23 Ci/mmol), [3H]uridine 5'-triphosphate (10 Ci/mol), and [3H]guanosine were obtained from Schwarz/Mann (Orangeburg, N.Y.). 32p, in 0.02 N HCI was obtained from ICN (Irvine, Calif.). Rifampin was purchased from Calbiochem (La Jolla, Calif.). Chloramphenicol (chloromycetin) sodium succinate was obtained from Parke-Davis (Detroit, Mich.). Isopropyl-38-D-thiogalactopyranoside was obtained from Sigma (St. Louis, Mo.). Acrylamide was purchased from Fisher Chemical Co. and was recrystallized from chloroform before use. Ethylenediacrylate was purchased from Borden Chemical Co. (Philadelphia, Pa.), and N,N,N',N'tetramethyl-ethylenediamine was purchased from Eastman Chemical Co. (Rochester, N.Y.). Orcinol and diphenylamine were both purchased from Sigma and were recrystallized before use. Determination of RNA, DNA, and protein. Aliquots of exponentially growing cultures were initially precipitated with 5% trichloroacetic acid. This step and all of the following steps were carried out at 4 C unless otherwise specified. The pellets were washed once by resuspension in 0.1 N NaOH and reprecipitation with 5% trichloroacetic acid. The pellets were then suspended in 0.2 N NaOH, and aliquots were removed and assayed for protein using a modified biuret procedure (25). The supernatant was made 0.5 N in base by the addition of KOH and incubated for 1 h at 37 C to hydrolyze the RNA. The remaining acid-insoluble material was precipitated by the addition of 1/10 volume of 6 N perchloric acid. The supernatant was neutralized with KOH and, after removal of the KCIO, precipitate, was analyzed for RNA content by the orcinol procedure (2). The precipitate was suspended in 10% perchloric acid and hydrolyzed for 30 min at 70 C to hydrolyze the deoxyribonucleic acid (DNA). The supernatant obtained by this hydrolysis was analyzed for DNA content by a modified diphenylamine procedure (11). Use of EDTA to prepare permeabilized cells. Cells were made permeable with ethylenediaminetetraacetate (EDTA) essentially as described by Rose et al. (27). Exponentially growing cultures at either 30 or 42 C were centrifuged briefly, suspended in 1/4 volume of 0.03 M tris(hydroxymethyl)aminomethane (Tris)-chloride (pH 7.3), and incubated with shaking for 2 min at the previous growth temperature; 0.1 M EDTA was added to give a final concentration of 1 mM, and the incubation was continued for an additional 2 min. Next, 3/4 volume of a concentrated growth medium was added (the concentration used was chosen that the final concentration, after dilution, was identical to the original growth medium). After 5 min of recovery at 30 or 42 C, the rate of RNA synthesis was measured by addso

J. BACTERIOL. ing 0. 1 pCi of [3H]uridine per ml and measuring the incorporation of 3H into acid-insoluble material. Control experiments showed that the 5-min recovery time was optimal in terms of both [3H]uridine uptake and sensitivity to rifampin. Cells treated in this manner are fully viable and will resume active growth after a brief lag but are fully permeable to [3H]uridine and various antibiotics (22). Preparation of toluenized cells and the RNA polymerase assay. Cells were treated with toluene essentially as described by Moses and Richardson (24) except that the buffer used for both toluenization and storage contained 50 mM Tris-chloride, pH 7.6, 2 mM P-mercaptoethanol, 2 mM KH2PO,, 100 mM KCI, 10 mM MgCl2, and 0.5 M sucrose. The toluenized cell suspension was stored frozen until ready to be assayed for RNA polymerase activity. The assay was carried out with 0.1-ml aliquots of the cell suspension diluted to give 15 to 45 isg of protein/0. 1 ml. Nucleoside triphosphates were added to give a final concentration of 0.25 mM adenosine 5'-triphosphate, guanosine 5'-triphosphate, and cytidine 5'-triphosphate and 0.15 mM uridine 5'-triphosphate with 0.25 ,uCi of [3H]uridine 5'-triphosphate per assay. Cells treated with toluene in this manner are not viable and cannot incorporate [3H]uridine into RNA. However, they are fully permeable and can be used to assay DNA or RNA polymerase in the presence of the appropriate nucleoside triphosphates. Electrophoretic analysis of the RNA. For analysis of the amount of rRNA and tRNA made at 30 and 42 C, cells grown on M9 medium plus glucose and Casamino Acids supplemented with 1.5 x 10-5 M unlabeled guanosine were labeled for 5, 10, or 15 min with 10 ACi of [3H]guanosine per ml. The RNA was isolated and purified by hot phenol extraction as described elsewhere (7). 14C-labeled marker RNA was prepared by adding 2 ;LCi of [14C]uridine to an exponentially growing culture of D10 containing 2 x 107 cells/ml. After a 10-fold increase in cell density, the cells were harvested and the RNA was purified by hot phenol extraction as above. Polyacrylamide gels (3.6% for rRNA and 6.0% for tRNA) were prepared as described by Corte et al. (6). The gels were equilibrated with the electrophoresis tank buffer (0.04 M Tris-0.02 M sodium acetate-0.2% sodium dodecyl sulfate-10% glycerol-1 mM EDTA0.2% glacial acetic acid, pH 7.2) for 30 min before being used. Aliquots of the 3H-labeled (short label) and "4C-labeled (long label) RNAs were mixed and applied to the gels in a total volume of 75 ,ul containing 0.04 M Tris, 0.02 M sodium acetate, 0.1% sodium dodecyl sulfate, 1 mM EDTA, 0.2% glacial acetic acid, 0.5 M sucrose, and 0.075% bromophenol blue. The 3.6% gels were run for 5 h at 5 mA/tube, and the 6.0% gels were run for 2 h at 5 mA/tube. In both cases the runs were carried out at room temperature. After electrophoresis, the 6.0% gels were frozen at -20 C, and the 3.6% gels were frozen with powdered dry ice. The gels were then sliced into 2-mm sections using a Hoefer model DE113 gel slicer (Hoefer Scientific Instruments, San Francisco, Calif.). The gel slices were each dissolved in 0.5 ml of the tissue solubilizer TS-1 (Research Products International,

VOL. 125, 1976 Elk Grove, Ill.) in capped scintillation vials at 37 C overnight. After cooling, 10 ml of toluene scintillation fluid was added, and the samples were counted in a refrigerated scintillation counter. This procedure gives optimal recovery of the counts and, using the appropriate channel settings, excellent resolution of the 3H and 14C counts. It is necessary to keep the samples at 4 C after adding the scintillation fluid, however, since otherwise a reaction occurs with the tissue solubilizer, which causes variable chemiluminescence. The '4C label was used both as a marker for stable RNA and as a measure of the of RNA from each gel. The 3H counts per minute in each gel were corrected for the recovery of 14C label in the same gel. However, that correction was small (usually less than 10 to 15%) compared to the observed differences between the 30 and 42 C gels. Other methods. The determination of MS1 levels has been described in a previous publication (5). Background values were determined for each sample using the charcoal treatment described by Gallant et al. (9). The 3-galactosidase assay was carried out essentially as described by Miller (23).

recovery

RESULTS Synthesis of RNA, DNA, and protein at 42 C. The accumulation of RNA and protein during growth at 42 C was measured chemically as described above. The kinetics of RNA and protein accumulation are shown in Fig. 1. Under those conditions the parental strain shows a continued exponential increase in both RNA and protein (5). 2S474 show an initial burst in RNA synthesis after the shift to 42 C; however, after 10 to 15 min all further accumulation of RNA ceases. Protein synthesis, on the other hand, continues at approximately the preshift rate. In the course of a 2-h incubation at 42 C, there is a 2.1-fold increase in protein content but only a 20% increase in RNA content-with essentially all of that increase occuring in the first 15 min. 2S139 shows a somewhat similar pattern. Net RNA accumulation slowly decreases at 42 C until it is reduced to 10 to 20% of the preshift rate, whereas protein synthesis continues at a slightly increased rate. The accumulation of DNA at 42 C was measured in a separate set of experiments and is shown in Table 1. In both cases, the accumulation of DNA observed at the restrictive temperature is similar to the accumulation of protein observed in Fig. 1, even though net accumulation of RNA has ceased. Effect of restrictive temperature on the rate of total RNA synthesis. To determine the effect of growth at the restrictive temperature on the rate of total RNA synthesis (stable plus unstable RNA), the rate of RNA synthesis was determined in cells which had been made permeable with EDTA. Cultures were grown either at

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30 C only or at 30 C followed by a 45-min incubation at 42 C. They were then centrifuged, treated with EDTA, and incubated with [3H]uridine as described above. For the cells grown at 42 C, both the EDTA treatment and the incubation were carried out at 42 C. The results of a typical experiment with 2S139 and 2S474 are shown in Fig. 2. In both cases the treatment with EDTA clearly renders the cells permeable to 20 gg of rifampin per ml at both 30 and 42 C. Furthermore, in experiments described elsewhere we have shown that the EDTA treatment does not appear to have any preferential effect on RNA synthesis at either temperature (manuscript in preparation). Thus, the uptake of [3H]uridine observed in Fig. 2 should be a fairly representative measure of the rate of RNA synthesis at the two temperatures. Based on these data, it appears that the rate of total RNA synthesis at 42 C decreases by a factor of approximately 2 in 2S139 and by a factor of approximately 10 in 2S474. However, in both strains there is an increase in the rate and extent of RNA turnover at 42 C (5). Thus, to truly measure the relative rates of RNA synthesis one needs to extrapolate to short enough labeling times so that the increased turnover at 42 C will have little effect on the uptake of label. The ratios of [3H]uridine incorporation at 30 and 42 C in the EDTA-treated cells after 1, 2, 5, and 10 min of labeling are shown in Table 2. From these data it appears that if one were to extrapolate to the shortest possible labeling time, the relative rates of RNA synthesis at 30 and 42 C would be about 7 for 2S474 and about 1 for 2S139. Thus, the rate of total RNA synthesis appears to be reduced approximately sevenfold in 2S474 at 42 C, but little, if any, in 2S139 at the restrictive temperature. Effect of the restrictive temperature on the synthesis of P-galactosidase. The parental strain and both mutant strains were tested for their ability to synthesize (-galatosidase messenger RNA (mRNA) at 42 C (Table 3). The rate of (3-galactosidase production at 30 C is proportional to the growth rate of the three strains at 30 C. When one looks at 3-galactosidase production at the restrictive temperature, it is clear that both 2S139 and 2S474 are inducible for f3-galactosidase. Thus the synthesis of at least one class of mRNA can continue at 42 C and that mRNA can be translated into functional protein. However, the rate of 13-galactosidase production is clearly reduced in both strains at 42 C. In each case, the reduction in fgalactosidase production is slightly greater than the observed reduction in RNA synthesis after very short labeling periods but agrees well

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FIG. 1. Effect of the temperature shift on RNA and protein synthesis. Exponentially growing cultures in glucose-salts media were shifted from 30 to 42 C at a cell density ofapproximately 108 cellslml. Aliquots were removed at the indicated times before and after the shift, and the RNA and protein contents were measured by the orcinol and biuret determinations as described. Symbols: 0, RNA; A, protein. TABLE 1. Accumulation of RNA and DNA during growth of 2S139 and 2S474 at 30 and 42 Ca Strain

Experimental

Turbidity at 540

RNA content

DNA content

nm

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+ 2hat42C 95 86.5 61 Relative increase at 42 C 1.46 1.05 1.48 a Cells were grown at 30 C to a final cell density of 1.3 x 108 cells/ml and then divided into two separate cultures. One culture was grown for another 2 h at 30 C, whereas the other was incubated for 2 h at 42 C. Aliquots (20 ml) were removed from the original culture just prior to the shift and from the subcultures after the 2-h incubation at the restrictive temperature. Each aliquot was analyzed for RNA and DNA content as described. Turbidity was measured in a Klett-Summerson colorimeter with a green filter.

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FIG. 2. Effect of the restrictive temperature on RNA synthesis in EDTA-treated cells. Cells were grown at 30 C in M9 medium plus glucose and Casamino Acids to a cell density of 108 cellslml. At this point one-half of the culture was shifted to 42 C, and the incubation was continued for 45 min at each temperature. Both cultures were then permeabilized with EDTA as described. In each case, 0.1 ,uCi of [3H]uridine per ml was added to the treated cultures, and the incorporation of the 3H into acid-insoluble material was measured by standard techniques. In the case of the cells grown at 42 C, both the incorporation study and each step of the permeabilization were also carried out at 42 C. Symbols: 0, 30 C; A, 42 C; 0, 30 C + 20 /g of rifampin per ml; V, 42 C + 20 pg of rifampin per ml.

Effect of the restrictive temperature on the synthesis of rRNA and tRNA. Previous studies, based on competition hybridization data, [3H]uridine incorporation at 30 C/ have suggested that both 2S139 and 2S474 were C at 42 [3H]uridine incorporation Strain deficient in the synthesis of rRNA at 42 C (5). 1 min 2 min 5 min 10 min To confirm this observation and to extend it to the synthesis of tRNA, the RNA produced after 1.76 1.14 1.39 0.94 2S139 5, 10, and 15 min of labeling at 30 and 42 C was 9.87 10.46 7.26 8.45 2S474 examined by polyacrylamide gel electrophorea The ratio of [3H]uridine uptake at 30 C/ sis as described above. Aliquots of the phenol[3H]uridine uptake at 42 C after 1, 2, 5, and 10 min of extracted RNA were electrophoresed on 3.6% labeling was calculated from the data presented in gels to determine the amount of 16S and 23S Fig. 2 for the EDTA-treated cells. rRNA present and on 6.0% gels to determine with the observed reduction in RNA synthesis the amount of tRNA present. In each case the After longer periods of labeling (Table 2). Thus samples were co-electrophoresed with an aliit would appear that the rate of 8-galactosidase quot of "4C-labeled stable RNA (preparation production at 42 C is approximately what one described above). The "4C-labeled RNA served would predict based on both the decreased rate both as a marker for migration of the stable of RNA synthesis and the increased rate of RNA species and as a measure of the recovery of each RNA species from the gel. The results turnover. TABLE 2. Ratio of [3H]uridine uptake at 42 and 30 C after various labeling timesa

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TABLE 3. Rate of ,¢galactosidase production at 30 and 42 Ca

Strain

Condition

D10 D10

30 C only 42 C for 45 min

2S139 2S139

30 C only 42 C for 45 min

Rate of Ratio ,-galactosidase (rate at production 42 C/rate (U/min per at 30 C) 108 cells)

11.9 16.0 0.818 0.459

1.34 0.56

2S474 30 C only 4.08 42 C for 45 min 0.433 0.11 2S474 a Cells were grown at 30 C on M9 medium plus glycerol and Casamino Acids to a cell density of 1 x 108 cells/ml. Each culture was then split into two aliquots, one of which was incubated at 30 C for 45 min and the other at 42 C. At this point 5 x 10-3 M isopropyl3- --thiogalactoside was added to each culture. Samples were removed at 5, 10, 15, and 20 min after the addition of the inducer and assayed for jSgalactosidase activity as described.

obtained with D10 after 15 min of labeling are shown in Fig. 3. Comparable results with 2S139 and 2S474 are shown in Fig. 4 and 5. As expected from the data in Fig. 2, the amount of [3H]uridine incorporated into both the rRNA and tRNA species at 42 C is reduced. However, it is also quite clear that the incorporation of [3H]uridine into rRNA is selectively depressed compared to the tRNA peak. This can best be seen by estimating the area under the rRNA and tRNA peaks and comparing them to the total trichloroacetic acid-precipitable counts (Fig. 6). When this is done it is clear that the composition of the RNA synthesized at 42 is radically different from that synthesized at 30 C. Whereas the percentage of tRNA remains about the same, the percentage of rRNA drops about 4-fold in the case of 2S139 and 5- to 10fold in the case of 2S474. Thus, in addition to a general decrease in RNA synthesis at 42 C, both strains show a selective decrease in rRNA synthesis and/or maturation. In the case of 2S474, it appears likely that it is rRNA synthesis that is affected at 42 C, since previous hybridization experiments with pulselabeled RNA have indicated a similar decrease in the percentage of rRNA made at 42 C (5). 2S139, on the other hand, presents somewhat more of a problem. The experiments described above showed that incubation at 42 C had little effect on the rate of total (stable plus unstable) RNA synthesis. Moreover, when one looks closely at the electrophoretic mobilities of the

RNA produced by D10 (Fig. 7) and 2S139 (Fig. 8) after 5, 10, and 15 min of labeling at 42 C, it is quite apparent that there is little, if any, conversion of p16S rRNA to ml6S rRNA in 2S139. No pl6S rRNA is detectable in D10 with labeling times longer than 5 min, whereas only p16S rRNA is found in 2S139 after 15 min of labeling. On the other hand, the hybridization studies (5) clearly indicate a preferential decrease in the amount of rRNA present even in pulse-labeled RNA. Presumably, then, the restrictive temperature may affect both rRNA synthesis and maturation to some extent in 2S139 (see below). Effect of the restrictive temperature on MS1 levels. We have previously reported (5) that both 2S139 and 2S474 show some elevation in MS1 levels at the restrictive temperature. Thus, the possibility existed that the cessation of rRNA synthesis at 42 C might be caused by the elevation in MS1 levels. To investigate this possibility further we investigated the effect of chloramphenicol on RNA synthesis and MS1 levels at the restrictive temperatures. Studies in other laboratories have shown that chloramphenicol reduces MS1 levels and stimulates RNA synthesis during amino acid starvation (4) and diauxie lag (9). However, when chloramphenicol was added to 2S139 at the restrictive temperature it had no effect on RNA synthesis-even though the concentration of MS1 fell to basal levels (Fig. 9). Clearly, then, the elevation in MS1 levels observed when 2S139 is shifted to the restrictive temperature is not enough in itself to explain the failure to accumulate rRNA. Similar results are obtained with 2S474, although the reduction in MS1 levels are not as dramatic (Fig. 10). Recovery from incubation at the restrictive temperature. Obviously, in both 2S139 and 2S474, some protein essential for rRNA synthesis and/or maturation is inactivated at 42 C. To determine whether the inactivation was reversible or not, the recovery of RNA synthesis after the cells were returned to 30 C was measured in the presence and absence of chloramphenicol (Fig. 11). 2S474 shows a very rapid recovery in RNA synthesis when it is returned to 30 C and the recovery is not affected by chloramphenicol. 2S139 shows a much slower recovery, but again the recovery is unaffected by chloramphenicol. Thus, in both cases, the inactivation observed at 42 C appears to be reversible. The cells are fully able to recover their ability to synthesize RNA even in the absence of any new protein synthesis. Effect of the restrictive temperature on RNA polymerase activity. To assess the effect

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FIG. 3. Gel electrophoresis ofthe RNA produced by D10 after 15 min of labeling at 30 and 42 C. Cells were grown at 30 C in M9 medium plus glucose, Casamino Acids, and 1.5 x 10-5 M carrier guanosine to a cell density of 12 x 108 cells/ml. At this point the cells were divided into two aliquots, one of which was incubated at 30 C for 45 min and the other at 42 C. At this point 10 puCi of [3H]guanosine per ml was added to each culture. After 15 min of labeling, aliquots were removed, and the RNA was extracted and purified as described. Electrophoresis was carried out on 3.6% polyacrylamide gel for 6.5 h (rRNA) and on 6.0% polyacrylamide gels for 2 h (tRNA) at 5 mA/tube. The gels were fractionated and counted as described. The positions of the mature 4S, 16S, and 23S RNAs were determined by co-electrophoresis with '4C-labeled marker RNA. (A) 30 C, 3.6% gel; (B) 30 C, 6.0% gel; (C) 42 C, 3.6% gel; (D) 42 C, 6.0% gel.

of temperature on RNA polymerase activity, cultures were grown either at 30 C only or at 30 C followed by a 45-min incubation at 42 C. In each case a toluene-treated cell suspension was prepared from these cultures and assayed for RNA polymerase activity at 30 C as described above. All of the assays in a given experiment were carried out with identical amounts of protein; thus, the observed incorporation of [3Hluridine 5'-triphosphate in each sample can be considered as a measure of the RNA polymerase activity per milligram of protein in that sample. A typical experiment with 2S139 and 2S474 is shown in Fig. 12. In 2S139 the RNA polymerase activity per milligram of protein is greater at 42 than it is at 30 C. This is similar to the effect one sees when the parental strain D10 is grown at 42 C. However, 2S474 actually

contains less RNA polymerase activity per milligram of protein at the high temperature (Fig. 12). This apparent loss in RNA polymerase activity is not due to a difference in cell recovery since both assays contained identical amounts of cell suspension. Nor is it due to a selective loss in RNA polymerase itself. The amount of the (3 subunits of RNA polymerase present in each cell suspension was determined as described elsewhere (manuscript in preparation). Based on the amount of f subunit present in each cell suspension, it appears that there is actually more RNA polymerase present in the toluene extract prepared from the 42 C cells, not less. Finally, there was no detectable difference in levels of ribonuclease activity in the two toluene cell suspensions. Thus, the apparent loss of RNA polymerase activity at 42 C is

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Conditions were the same as described in the legend to Fig. 3 except that the labeling at 42 C was done with 20 ,ICi of[3H]guanosine per ml in the absence of any added carrier (final concentration, 10-6 M). (A) 30 C, 3.6% gel; (B) 30 C, 6.0% gel; (C) 42 C, 3.6% gel; (D) 42 C, 6.0% gel.

elevated temperature (42 C) -will be minimized. Thus, we feel this method should provide an accurate estimate of the relative rate of although the cause of that inactivation remains total RNA synthesis at the two temperatures. The estimate of the percentage of rRNA and to be determined. tRNA made on the basis of the counts recovered in two different gels might be expected to lead DISCUSSION to some problems if there were a difference in Evaluation of the experimental approach. the recovery of the RNAs from one gel to anThe effect of temperature on the rate of total other. For example, if there were an aggregaRNA synthesis in these strains was evaluated tion of rRNA's so that they did not enter the using cells permeabilized with EDTA. We have 3.6% gel one would obtain a systematic undershown elsewhere (manuscript in preparation) estimate of the percentage of rRNA present in that EDTA has no preferential effect on RNA the labeled RNA. However, one needs to keep synthesis at either of the two temperatures in mind that the important comparison here is tested. From the data in Fig. 2, the cells are not between the percentage of rRNA and tRNA clearly fully permeable to low levels ofrifampin which accumulate at any one temperature, but at both temperatures. Furthermore, by extrap- rather between the percentage of rRNA or olating to the shortest pulse-labeling times (Ta- tRNA which accumulate at 30 C compared to ble 2), the effect of any increase in RNA turn- the percentage of the same RNA which accuover-either caused by the EDTA (30) or by the mulates at 42 C. Furthermore, the recovery of not due to the unmasking of ribonuclease activity. Clearly, then, there appears to be a reduction in the activity of RNA polymerase at 42 C,

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31 Is 11 3: flCIN MAD RNA FIG. 5. Gel electrophoresis of the produced by 2S474 after 15 min of labeling at 30 and 42 C. The conditions were the same as described in the legend to Fig. 4. (A) 30 C, 3.6% gel; (B) 30 C, 6.0% gel; (C) 42 C, 3.6% gel; (D) 42 C, 6.0% gel. the RNAs from each gel was standardized by with the EDTA-permeabilized cells it appears co-electrophoresis with known quantities of 14C0 that the rate of total RNA synthesis is de. labeled marker RNAs. In each case the 3H creased approximately sevenfold at the restriccounts obtained in the peak fractions were cor- tive temperature. The data with 3-galactosidrected for the recovery of the 14C counts in the ase suggest that mRNA synthesis is affected to same gel. Moreover, this correction was invari- about the same extent. Furthermore, the data ably small (10 to 15%) compared to the large obtained from the 6.0% polyacrylamide gels differences observed in the 3H-labeled rRNA show that the percentage of tRNA synthesized present at the two temperatures. is unchanged at 42 C, suggesting that tRNA We cannot exclude the possibility that the 4S synthesis is also decreased to about the same peak in the 6% gels might contain some par- extent as total RNA synthesis. However, the tially degraded rRNA as well as tRNA. Al- gels also show a 5- to 10-fold decrease in the though the close agreement between the gel percentage of rRNA made at 42 as compared to electrophoresis and competition hybridization 30 C. This would amount to a 35- to 70-fold (5) experiments make this unlikely, we also reduction in the rate of rRNA synthesis. Hence, examined this question by labeling the cells clearly rRNA synthesis and/or maturation is simultaneously with [methyl-3H]methionine preferentially affected at the restrictive temand [14C]uridine (unpublished data). Based on perature. The fact that one sees a similar dethe methionine to uridine ratio of the 4S peaks, crease in the percentage rRNA in pulse-labeled it appears likely that all of the 4S peak is tRNA RNA analyzed by hybridization (5) suggests that the primary effect is on the synthesis ofthe in 2S139 and at least 70% of it is in 2S474. What is the nature of the temperature-sen- rRNA, not on the maturation. This is also sugsitive lesion in 2S474? From the experiments gested by the fact that the little rRNA which is I1

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TINE r 1 EETIU ( NIN ) FIG. 6. Composition of the RNA synthesized by D10, 2S139, and 2S474 at 30 and 42 C. The incorporation of [3H]uridine into the total acid-insoluble fraction and into rRNA and tRNA was measured in the experiments described in the legends to Fig. 3 to 5. Symbols: 0, total acid-insoluble radioactivity; A, 16S + 23S rRNA; 0, 4S RNA.

found in the gels at 42 C seems to migrate in mulation of MS1 observed during amino acid the position expected for m23S and ml6S (al- starvation (4, 15) is associated with a coordinate though the small number of counts present block in the synthesis of all stable classes of would make it difficult to detect small changes RNA. Secondly, the temperature-sensitive lein mobility). sion in 2S474 shows a negligible frequency of We do see an elevation of MS1 levels at 42 C. cotransduction with the argA locus and the rif Furthermore, chloramphenicol, which does not locus, which are closely linked to the relA and stimulate rRNA synthesis, also has no dra- relC loci, respectively. Thus, the temperaturematic effect on MS1 levels in this mutant. How- sensitive lesion is most likely not in either the ever, we still do not think that the elevated relA or relC locus. We have not checked this MS1 levels are the cause of the observed defect mutant phenotype for cotransduction with the in rRNA synthesis at 42 C. In the first place, spoT locus. However, we do not feel that this we saw a preferential reduction in rRNA syn- mutant is a temperature-sensitive spoT muthesis in this mutant at 42 C, whereas the accu- tant, since even completely spoT- mutants do

MUTANTS IN rRNA METABOLISM

VOL. 125, 1976

1067

4~~~~~~~~~~4 X IS

S

1,

lS

23

x ~ U

x

U

fRRCTIIN UBEER FIG. 7. Maturation of rRNA in D10 at 42 C. The conditions were the same as described in the legend to Fig. 3. Symbols: -, 15 min of labeling, - -, 10 min of labeling; , 5 min of labeling.

not show significant elevation of ppGpp levels in the absence of amino acid starvation (23). The experiments with toluenized cells suggest RNA polymerase or some auxiliary enzyme required for RNA synthesis may be iliactivated at 42 C. This is probably not due to a simple heat inactivation of the RNA polymerase itself, since the mutant enzyme is not detectably more temperature sensitive than the wild-type enzyme in vitro (data not shown). This alteration presumably results in a generalized decrease in the ability to synthesize all classes of RNA, but with specific preferential decrease in the ability to synthesize rRNA. The nature of this lesion is unknown at present. We have recently described another mutant strain of E. coli with very similar properties (manuscript in preparation). These two mutants should prove useful in delineating the role of RNA polymerase in the synthesis of rRNA. What is the nature of the temperature-sensitive lesion in 2S139? The situation with 2S139 is somewhat less clear. As discussed above, it

would appear that both rRNA synthesis and maturation might be affected to some extent in this strain at the restrictive temperature. Again MS1 levels are observed to increase in this strain at the restrictive temperature. Here, however, the increased MS1 levels are clearly not the cause of the defects in rRNA metabolism. Not only is the effect specific for rRNA, but chloramphenicol reduces MS1 to basal levels in this strain at 42 C without any detectable effect on RNA synthesis. There appears to be no demonstrable alteration of RNA polymerase either in vivo or in vitro at the restrictive temperature. Thus, the mechanism of this temperature-sensitive lesion is unknown, but it is clearly complex. Since both synthesis and maturation of rRNA appear to be affected at the restrictive temperature, whereas protein synthesis continues unabated, this mutant should provide a useful system for studying the possible relationships between the synthesis of ribosomal protein and the synthesis and maturation of rRNA.

1068

JACKSON AND CHANEY

J. BACTERIOL.

Ibb

gJZ

Ari

11

IS

21

U3

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FRFCTION NOW FIG. 8. Maturation of rRNA in 2S139 at 42 C. The conditions were the same as described in the legend to 5 min of labeling. Fig. 4. Symbols -, 15 min of labeling; ---, 10 min of labeling;

MUTANTS IN rRNA METABOLISM

VOL. 125, 1976

P%

1069

n

U

uI'4

K x

U

L

z Ft -J

w

ut

I

rb

V

EO

3

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la

I

In

211

NKIN )

FIG. 9. Effect of chloramphenicol on RNA synthesis and MS1 levels in 2S139. Cells were grown on lowphosphate medium (2) supplemented with glucose and all 20 amino acids at 5 mglml. Growth was at 30 C to a final cell density of 1.5 x 108 cellslml, at which point the cells were shifted to 42 C. For the measurement of RNA synthesis under the restrictive conditions, the growth medium was supplemented with 5 x 10-5 M carrier uridine, and 0.5 ,uCi of [3H]uridine per ml was added at a cell density of 7 x 107 cellslml. At "C indicated intervals, aliquots were removed and 3H-labeled acid-insoluble material was measured by standard techniques. MS1 levels were measured as described previously (5). Chloramphenicol was added at a final concentration of 100 Aglml. Symbols: 0 O, RNA synthesis, MSJ levels are indicated by bars at the time of

sampling.

1070

JACKSON AND CHANEY

J. BACTERIOL.

'U In U x

In

32 ul

jLa

321

E

In

16

em

a

U

U

3

1a TINE ( KIN )

Ise

i

211

FIG. 10. Effect of chloramphenicol on RNA synthesis and MS1 levels in 2S474. The conditions were the same as described in the legend to Fig. 9. Symbols: O-O, RNA synthesis. MSJ levels are indicated by bars at the time of sampling.

MUTANTS IN rRNA METABOLISM

VOL. 125, 1976

P1

A

-

ILIC 0I

*

u

~

1

1071

~~

61

~ NOm~ DDITION

121

1 TINE Or REGER

NO

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RDDITIN

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FIG. 11. Effect of chloramphenicol on the recovery of 2S139 and 2S474 after growth at 42 C. Cells were grown on M9 medium supplemented with glucose, Casamino Acidls, and 5 x 10-5 carrier uridine at 30 C to a cell density of 7 x 107 cellslml. At this point 0.5 iLCi of [3H]uridine per ml was added to the culture. After 20 min at 30 C the cells were shifted to 42 C for 45 min. At this point the cultures were divided into two aliquots. Both were incubated at 30 C, but 1 00 pg of chloramphenicol per ml was added to one. Incorporation of 3H into acid-insoluble material was measured by standard techniques. Symbols: O, no addition; A\, 100 pg of chloramphenicol per ml added.

1072

JACKSON AND CHANEY

J. BACTERIOL.

16 N

U x x

12

e1 W

a

9

V .j

4I

20

II

TINE ( MIN ) FIG. 12. RNA polymerase activity in toluenized cell suspensions. Cultures (80 ml) of2S139 or 2S474 were grown on M9 medium plus glucose and Casamino Acids at 30 C to a final cell density of1.5 x 10 cells/ml. At this point the cultures were divided into two aliquots, one of which was incubated at 30 and the other at 42 C. After 45 min the cells were pelleted, and toluene-treated cell suspensions were prepared and assayed as described. Each assay was carried out at 30 C for the indicated length of time with 45 ug of total proteinlO.1 ml. Symbols: 0, 2S139 grown at 30 C; A, 2S139 grown at 42 C; O, 2S474 grown at 30 C; A, 2S474 grown at 42 C. ACKNOWLEDGMENTS This work was supported by grant number GB-36974 from the National Science Foundation, Public Health Service grant number 1-RO1-CA15697-01 from the National Cancer Institute, and institutional grant number IN-15-N from the American Cancer Society to the University of North Carolina Medical School. LITERATURE CITED 1. Adams, M. H. 1966. Bacteriophages, p. 446. Interscience Publishers, New York. 2. Ashwell, G. 1957. Colorimetric analysis of sugars, p. 87-90. In S. P. Colowick and N. 0. Kaplan (ed.), Methods in enzymology, vol. 3. Academic Press Inc., New York. 3. Babinet, C. 1970. A mutation which affects the resistance of E. coli to rifampin. Lepetit Colloq. Biol. Med. 1:37-45. 4. Cashel, M. 1969. The control of RNA synthesis in E. coli. IV. Relevance of unusual phosphorylated compounds from amino acid-starved stringent strains. J. Biol. Chem. 244:3133-3141. 5. Chaney, S. G., and D. Schlessinger. 1975. Escherichia coli mutants deficient in RNA accumulation at high temperature. Biochim. Biophys. Acta 378:80-91. 6. Corte, G., D. Schlessinger, D. Longo, and P. Venkov.

7.

8. 9. 10.

1971. Transformation of 17 S to 16 S ribosomal RNA using ribonuclease II of Escherichia coli. J. Mol. Biol. 60:325-338. Craig, E., K. Cremer, and D. Schiessinger. 1972. Metabolism of T4 messenger RNA, host messenger RNA, and ribosomal RNA in T4-infected E. coli B. J. Mol. Biol. 71:701-715. Fiil, N., and J. D. Friesen. 1968. Isolation of "relaxed" mutants of Escherichia coli. J. Bacteriol. 95:729-731. Gallant, J., G. Morgason, and B. Finch. 1972. On the turnover of ppGpp in Escherichia coli. J. Biol. Chem. 247:6055-6058. Gesteland, R. F. 1966. Isolation and characterization of ribonuclease I mutants of E. coli. J. Mol. Biol. 16:67-

84. 11. Giles, K. W., and A. Myers. 1956. An improved diphenylamine method for the estimation of DNA. Nature (London) 206:93. 12. Harschman, R. B., and H. Yamasaki. 1971. Formation of ppGpp in a relaxed and stringent strain of E. coli during diauxie lag. Biochemistry 10:3980-3982. 13. Haseltine, W. A., and R. Block. 1973. Synthesis of guanosine tetra- and pentaphosphate requires the presence of a codon-specific, uncharged transfer RNA in the acceptor site of ribosomes. Proc. Natl. Acad. Sci. U.S.A. 70:1564-1568. 14. Haseltine, W. A., R. Block, W. Gilbert, and K. Weber.

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16.

17.

18.

19.

20. 21.

22.

1972. MSI and MSII made on ribosome in idling step of protein synthesis. Nature (London) 238:381-384. Jackunas, S. R., M. Nomura, and J Davies. 1975. Genetics of bacterial ribosomes, p. 333-368. In M. Nomura, A. Tissieres, and P. Lengyel, (ed.), Ribosomes. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Jacobson, A., and D. Gillespie. 1970. An RNA polymerase mutant defective in ATP initiations Cold Spring Harbor Symp. Quant. Biol. 35:85-93. Khan, S. R., and H. Yamasaki. 1974. Correlation between guanosine tetraphosphate accumulation and degree of amino acid control of RNA accumulation during nutritionally slowed growth in E. coli. Biochemistry 13:2785-2788. Khesin, R. B., S. Z. Mindlin, Z. M. Gorlenko, and T. A. Ilyina. 1968. Temperature sensitive mutations affecting RNA synthesis in Escherichia coli. Mol. Gen. Genet. 103:194-208. Khesin, R. B., M. F. Shemyokin, Z. M. Gorlenko, S. Z. Mindlin, and T. S. Ilyina. 1969. Studies on the RNA polymerase in Escherichia coli K12 using the mutation affecting its activity. J. Mol. Biol. 42:401-419. Lamer, T., and J. Gallant. 1974. spoT, a new genetic locus involved in the stringent response in E. coli. Cell 1:27-30. Lazzarini, R. A., M. Cashel, and J. Gallant. 1971. On the regulation of guanosine tetraphosphate levels iD stringent and relaxed strains ofE. coli. J. Biol. Chem. 246:4381-4385. Leive, L. 1965. Actinomycin sensitivity in Escherichia

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25. 26. 27.

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coli produced by EDTA. Biochem. Biophys. Res. Commun. 18:13-17. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Moses, R. E., and C. C. Richardson. 1970. Replication and repair of DNA in cells of E. coli treated with toluene. Proc. Natl. Acad. Sci. U.S.A. 67:647-681. Munkos, K. D., and F. M. Richards. 1965. The purification and properties of neurospora malate dehydrogenase. Arch. Biochem. Biophys. 109:466479. Patterson, D., M. Weinstein, S. Marshall, and D. Gillespie. 1971. A new RNA synthesis mutant of E. coli. Biochem. Genet. 5:563-578. Rose, J. K., R. D. Mosteller, and C. Yanofsky. 1970. Tryptophan messenger RNA elongation rates and steady-state levels of tryptophan operon enzymes under various growth conditions. J. Mol. Biol. 51:541550. Schlessinger, D., and D. Apirion. 1969. Escherichia coli ribosomes: recent developments. Annu. Rev. Microbiol. 23:387426. Ryan, A. M., and E. Borek. 1971. The relaxed control phenomenon. Prog. Nucleic Acid Res. Mol. Biol. 11:193-228. Yuan, D. and V. Shen. 1975. Stability of ribosomal and transfer ribonucleic acid in Escherichia coli B/r after treatment with ethylenedinitrilotetraacetic acid and rifampin. J. Bacteriol. 122:425-432. Yura, T., K. Igarishi, and K. Mataukata. 1970. Temperature-sensitive RNA polymerase mutants of Escherichia coli. Lepetit Colloq. Biol. Med. 1:71-89.