Some effects of streptomycin on RNA metabolism in Escherichia coli

J. Mol. Biol. (1964) 8, 749-767

Some Effects of Streptomycin on RNA Metabolism in Escherichia coli DONALD T. DUBIN Department of Bact eriology and I mmunology, H arvard M edical School Boston 15, jlIassuchusetts, U.S.A .

(Received 18 December 1963, and in revised form 11 February 1964) Streptomycin is known to allow considerable ribonucleic acid synthesis even after it has stopped protein synthesis in sensitive cells. This "streptomycin RNA" has now been shown to resemble chloramphenicol RNA in that (1) it has a relatively normal size distribution when purified and fracti onated by means of sucr ose density-gradient centrifugation; (2) a portion of it is incorporated into abnormally slowly sedimenting particles; and (3) a portion (perhaps the same portion as in (2» is unstable both in cells and in cell extracts. Streptomycin can also cause a net stimulation of RNA synthesis, and at low drug concentrations this stimulation precedes detectable slowing of protein synthesis. The early stimulation has been shown to involve each of the major centrifugally-separable classes of normal RNA as well as m essenger RNA. In addition, the ribosomal components in this case are in corporated into normal ribosomal subunits. Similar results were obtained using very low concentrations of chloramphenicol. It is proposed that the stimulation of RNA synthesis by both drugs may be the result of inhibition of protein synthesis too sligh t t o be detected directly. While the effects of chloramphenicol and streptomycin on RNA synt hesis thus appear quite similar, t heir effects on RNA stabilit y d iffer significantly. Strep tomycin-t reated cells undergo a generalized RNA breakdown which cont r as ts with the limited breakdown to which both chlorampheni col RNA and st re p to mycin RNA are susceptible. The present findings are discussed with reference to the regulation of RNA synt hesis as well as to the possible mechanisms of action of streptomycin.

1. Introduction Rec ent reports on the action of STMt have described several effects on RNA metabolism in sensitive bacteria. The synthesis of RNA proceeds in the presence of STM after the cessation of protein synthesis (Hahn & Ciak, 1959 ; Anand & Davis, 1960). Several laboratories have reported that this RNA is abnormally rich in slowlysedimenting components (Eaton & Caffrey, 1961; White & Fl ak s, 1962; Wolfe & Hahn, 1963). More recently, an actual stimulation of RNA synthesis by STM has been demonstrated (Dubin, Haucock & Davis, 1963). At low drug concentrat ions this stimulation was detectable as early as the first indication of the membrane damage suffered by the cells, and slightly before loss of viability and slowing of protein synthesis. STM has also been shown to trigger an extensive, although late, degradation of cellular RNA (Dubin & Davis, 1962).

t Abb re via.tions u sed: STM = streptomycin ; CM = chloramphenicol ; ST M R NA = st rep to my cin r ib onucleic acid; CM RNA = chloramphen icol ribonucleic a cid; T CA = trich loroacetic a cid; M-RNA = m essenger ribonucleic ac id ; R·RNA = ribosomal ribonucle ic a cid ; S -R NA = so lu b le ribonucleic acid. 749

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D. T. DUBIN

The present studies were aimed at obtaining a more complete picture of the alterations of RNA metabolism in STM-treated cells. In particular, the nature of the RNA synthesized during exposure to STM and the stability of this RNA and pre-existing RNA were investigated. Since many of the observed effects resembled certain wellstudied effects of OM, direct comparisons were made between the two drugs.

2. Materials and Methods Growth of cells. Unless stated otherwise, Escherichia coli strain ML35 was grown at 37°C with aeration in a minimal phosphate-buffered medium ("A /K o.3" ) supplemented with 0·2% glucose, as previously described (Dubin et al., 1963). Nucleic acid precursors and leucine were added as indicated at the following concentrations: leucine, 25 to 35 fLg/ml.; guanine, 25 to 35 fLg/ml.; and uracil (except in [14C]uracil "pulse" experiments), 10 fLg/ml. Growth was followed by reading turbidity in a Lumetron colorimeter. A reading of 0·1 represented approximately 0·09 mg of cells per ml. (Here, as below, cell weight refers to dry weight.) Doubling times of cultures growing exponentially at 37°C were approximately 40 min in the absence of leucine, and 45 min in its presence (see Results). Radioactive materials. [14C]guanine (22,3 fLo/mg), [14C]leucine (143 fLo/mg), [14C]uracil (two lots, 280 fLo/mg and 91 fLo/mg) and [3H]uracil (31,000 fLo/mg) were obtained from New England Nuclear Corp., Boston, Mass. [3ZP]phosphate was obtained from Iso/Serve Corp., Cambridge, Mass. The synthesis of protein, RNA and DNA was followed by measuring the incorporation of 14C·labeled precursors into TCA precipitates as previously described (Dubin et al., 1963). In exponentially growing cultures, 12 to 14% of the 14C incorporated from guanine or uracil was in DNA. Total protein was measured by the method of Lowry, Rosebrough, Farr & Randall (1951), using bovine serum albumin as standard. Total RNA was measured by the orcinol reaction according to Mejbaum (1939), using as standard thymus sodium ribonucleate (Nutritional Biochemicals Corp., Cleveland, Ohio) of known phosphate content. Nucleic acid phosphate was measured by a modification (Ames & Dubin, 1960) of the method of Chen, Toribara & Warner (1956), after ashing according to King (1932). The phosphorus content of pure RNA was taken as 9'6%. Values obtained were in good agreement with estimates based on [3ZP]phosphate incorporation. Preparation of cell extracts. Cells were rapidly chilled by pouring cultures onto approximately 0'5 vol. of crushed ice. Carrier and labeled cells, in a ratio of 12 to 20: 1, were harvested together by centrifugation in the cold. The cells were resuspended evenly in tris-HCI, pH 7,4, 5 x 10-3 M ("tris buffer"), containing 5 x 10-3 M.MgClz' This suspension was divided into two approximately equal parts, each of which was centrifuged separately. The cells to be used for the crude extract were washed once with tris buffer containing 10- 4 M·MgClz' and then processed in the cold essentially as described by Tissieres, Watson, Schlessinger & Hollingworth (1959). The pellet was ground with alumina (Alcoa, bacteriological grade A-305) and then extracted with tria buffer containing 10- 4 M-MgClz and 5fLg/ml. of DNase (Worthington Biochemicals, Freehold, N.J.). Alumina and cell debris were removed by centrifugation for 20 and then 30 min at 15,000 g. In some experiments the extracts were finally dialysed for 8 hr in the cold against 100 vol. of tris buffer containing 10- 4 M-MgClz. Dialysed extracts contained smaller amounts of slowly sedimenting non-acid-precipitable material than non-dialysed extracts, and hence yielded more clearly defined 4 s RNA peaks; dialysed extracts were otherwise indistinguishable from undialysed extracts. The extracts were adjusted to contain 3 to 4 mg of nucleic acid per ml., less than 2% of which was DNA (measured as acid-precipitable radioactive material after alkaline hydrolysis (Dubin et al., 1963)). Yields of RNA, based on 14C in TCA precipitates of samples of total culture, ranged from 50 to 80%. RNA was prepared by a slight modification of a method kindly suggested by Dr. K. Asano (personal communication). The method was originally designed to obtain bacterial RNA in good yield and with a minimum of degradation of M-RNA or of association between M-RNA and R-RNA. In the present experiments yields of total RNA (calculated as above) were 50 to 60%; hence since some purely mechanical loss was unavoidable, the purified

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RNA obtained was considered representative of the cellula r RNA. In addition , an exper im ent in which cells were harvested after a 30·sec pulse of [HC]uracil (se e Monier, Naono, Hay es, Hay es & Gros, 1962) confirmed the finding (Asano, 1963) t hat the method y ields undegraded (i.e. polydisperse) M·RNA, unassociated wi th R-RNA . The procedure was as foll ow s: after the ini tial wash the pelleted cells were r esu sp ended, at a d en sity of approximat ely 20 m g jrnl. , in sodium acetate buffer, pH 5'1, 0'0075 M , co ntain ing 7 m g jrnl . of bent oni t e (F ish er Scientific, U .S .P ., prep ared according to F raenk el -Conra t , Sin ger & Tsu gita (1961)) ; 20 mg/ml. of sodium dodecyl sulfate (recryst a lli zed according to Crestfleld, Sm it h & All en, 1955); and 0'005 xr-s odium EDTA. This mi x ture was held a t 37 °C, with periodic st irring, for 15 min a nd then sha ke n wi th a n eq ua l volume of freshly r edistilled , wa ter -sa t urated phenol (Mallinckrod t , liquefied ) for 20 m in at room temp erature . The phases were separated by centr ifuga t ion at 15,000 g for 20 min, after which the phenolic phase was removed and re-extracted with a n eq ual vo lume of water. The com bine d aqueous phases were then r e-extrac t ed three t im es with fresh phenol. Approximately 0·1 vol. of 2 M-sodium acetate, pH 5'1 , was adde d and the nucleic a cid was precipitated at O°C with 2 vol. of et ha n ol. After centr ifugation, t he nucleic ac id was taken up in sodium acetate buffer, pH 5'1, 0,01111, conta in ing 0·05 M-NaCI ("acetate-NaCI"), and then reprecipitated with ethanol. The precipi tate was then treated for 10 min at room temperature with lOJkg/ml. of DNase in acet ate-NaCI containing 10- 3 M.MgCI 2 • This treatment markedly reduced the viscosity of the preparation and increased the ease with which subsequent ethanol precipitates cou ld b e redissolved, although m ost of the DNA remained acid- and ethanol-precipitable, and sed imen t ed only slightly slowe r t h an 16 s RNA. After two more e th a nol precipitat ion s , the material wa s a gain treated wi th DNase and then reprecipitated twice more with eth a no l. I t was then dissolv ed in acetat e-NaCI to make a final con cen t ra t ion of 3 to 4 mg of RNA /ml. , and centrifuged for 20 min at 15,000 g in the cold . The final supernatant solu t ion containe d approximat ely 5 % of its total coun ts as DNA, which s ed imen te d w ith the 4 s RNA. Sucrose gradients . Linear 5 t o 20 % sucrose gra d ients were made u p as previously d escrib ed (Britten & Roberts, 1960) using t ris buffer containing 10- 4 M·l\'1gCI 2 for crude ext racts, a nd aceteto-NaOl for purified RNA. Sa m p les of 0·1 to 0·2 ml. wer e loaded onto 4· 8 ml. gr ad ients, which were then run in a swinging buck et (SW39) head in a Sp inc o m od el L ul tracentrifuge set at O°F. Samples of t h ree drops eac h we re t hen collec ted , diluted with 1 ml. of water for readin g absorbancy at 260 mp. (us ing a B eckman DU spectrophotomet er) and then poured into scin t illation bottles contai ni ng 15 m l. of Bray's solvent (Bray, 1960). 3H and HC were coun ted sim u lt a ne ous ly in a N u cle a r Chicago scintillation counter adjusted to g ive efficienc ies for 3H of approximately 3 % in Cha nne l I and less than 0·03 % in Channel II; and efficienc ies for HC of approxima tely 20% in Channel II and 4% in Channell. Variations of s ucrose ov er t he range of co ncentrations us ed did not alter the counting rate, and r esults were quite reproducibl e in a ser ies of samples counted on the same day (as was done for each g roup of t hree gradients). However, there was considerable variability in counting efficienc y fr om day to day. D espite an attempt to correct for this by count in g standards with eac h ser ies, it was felt that no more than rough comparisons of spe cific radioactivities cou ld be made between gradients run on separate days. Since actual corrected sedimentation vel ocities were not obtained , S- values have been us ed throughout only as convenient labels for the major centrifugally sep a ra ble classes of ribosomal sub unit s and of RNA in E. coli , as described in ea r lie r work (cf. 'I'issieres et al ., 1959 ; Kurland, 1960).

3. Results (a) General characteristics of the stimulation of net RNA syn thesis by streptomycin Th e net synthesis of RNA was studied by allowing cells to incorp orat e radioacti ve nucleic acid precursors over relatively long time periods (30 to 90 minutes). The cells were grown in the presence of a sufficient concentration of the pr ecur sor to prevent depl etion during the course of an experiment. Labeled comp ounds were added to both control and drug-treated cultures at the same time as drug was added to the 49

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752

latter. In most experiments unlabeled leucine was also added to the growth medium so that the experiments might be comparable to those in which the incorporation of [14C]leucinewas studied (see next section and Dubin et al. , 1963). The leucine , however, had an unexpected effect: it caused slowing of the basal rate of RNA synthesis relative to protein content, as well as slowing of the over-all growth rate, in the control cultures. The mechanism of this inhibitory action of leucine was not investigated in detail. Whatever the mechanism, however, the end result was to magnify the early stimulation of RNA synthesis by both STM and CM, as described below.

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FIG. 1. Effects oj eeoeral concentrations oj 8treptomy cin on net RNA BYnthesi8. Cells were grown in medium A :K o.s supplemented with leucine and guanine. At a turbidity of 0·26 (zero time), portions were added to equal volumes of warm medium containing [UClguanine, 0·014/ka/mI., plus STM at the (final) concentrations indicated (in /kg/mI.). Incorporation of radioactivity into RNA was followed by counting cold TCA precipitates lIS described in the text. Results are expressed lIS percentage of the parallel control culture.

Figure 1 illustrates the pattern of net RNA synthesis in cells exposed to varying concentrations of STM in the presence of leucine and [14C]guanine. [14C]uracil gave similar results. The higher the drug concentration, the earlier and more marked, but the less prolonged, was the early stimulation. Net RNA synthesis continued for 60 to 90 minutes in the various treated cultures. Beyond this time the incorporated radioactivity actually decreased, owing to concurrent RNA breakdown (cf. Dubin & Davis, 1962) in all the treated cultures except the one exposed to 1000 p,gJml. of STM; this high concentration itself prevents RNA breakdown (Feingold & Davis, 1962). As was previously found for low levels of STM (Dubin et al., 1963), the stimulation of [14C]guanine incorporation at all these drug levels was due to an acceleration of net RNA synthesis. Although DNA, like RNA, continued to be synthesized even after the cessation of protein synthesis, no stimulation of DNA synthesis was observed. In the experiments reported in this paper, both "high" (1000 p,gJml.) and "low" (20 to 30 p,gJml.) concentrations of STM were used. A high concentration of the drug caused over 90% killing and marked slowing of protein synthesis (to less than 5%

STREPTOMYCIN AND RNA METABOLISM IN E. O O L I

753

of normal) within five minutes. With "low concentrations" of STM, there was generally a lag of approximately 10 minutes before the stimulation of RNA syn the sis, and another lag of approximately five minutes before the onsets of slowing of protein syn thesis and loss of viability. As indi cated above, leucine slowed the rate of RNA synthesis in control cultures and at the same time magnified the stimulat ion of RNA synthesis in S'I'Mvtreat ed cultures. When the growth medium was supplement ed in addition with a mixture of nineteen ot her amino acids, both these effects ofleucine were more t ha n counteract ed .

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0 FIG . 2. Effect of am i7W acids on the stimulation of R NA synthesis by streptomycin. Cells were grown for approximately three generations in on e or t he other of t wo growth m edia: A'K o., supplem ented wit h uracil and leucine, or the same medium sup plemented in addit ion with o·}",mol e/mI. of the other nineteen naturall y -occurring amino a cid s. At a t u rbid ity of 0·2 (zero time) portions of each culture were added to equa l volumes of fres h, warm m ed ium containing [l' C]uracil, 0·018 p.c /ml. , plus or minus ST~I to give a final concentration of 30 ",g/ml. Incorporation of radioactivity into RNA wa s followed by counting cold T CA precipitates, as d escribed in the text. Cultures supplemented with 20 amino a cids: - - • - - • - - , control; - - . - - . - - , STM. Cultures supplem ented with leucine: - 0 -- 0-, control; -6 - - 6 - , STM.

As shown in Fig. 2, the rate of RNA synthesis in untreated cells was stimulated over threefold by the amino acid mixture, and there was no superimposed stimulation by STM. Cells growing in minimal medium with no added amino a cids synt hesized RNA at an intermediate rate, and also showed an intermediate degree of susceptibility to the stimulation of RNA synthesis. Similar r esults were obtained for CM, in agreement with earlier studies on this drug (Kurland & Maalee, 1962). (b) Timing of the stimulation of net RNA synthesis by low levels of chloramphenicol

Kurland & MaaliJe (1962) have provided evidence that the st imulat ion of RNA syn thesi s by CM (Fraenkel & Neidhardt, 1961) is secondary to the inhibitory effect of this drug on protein synthesis. They ha ve suggest ed that S-RNA un est erified with

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amino acids ("uncharged" S-RNA) "represses" RNA synthesis; and that when CM slows protein synthesis, charged S-RNA accumulates at the expense of uncharged S-RNA, thus releasing this "repression". Such a regulatory function for S-RNA has also been inferred from genetic studies (Stent & Brenner, 1961) and from studies on in vitro RNA synthesis (Tissieres, Bourgeois & Gros, 1963). Since STM (Flaks, Cox & White, 1962; Speyer, Lengyel & Basilio, 1962; Davies, 1963) like CM (Gale & Folkes, 1955) has a direct effect on the bacterial protein-synthesizing system, it seemed reasonable to try to explain the STM-induced stimulation of RNA synthesis along the same lines. However, with low levels of STM the stimulation of RNA synthesis preceded detectable slowing of protein synthesis (Dubin et al., 1963). This finding

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FIG. 3. Effects oj low levels oj chloramphenicol on RNA and protein synthesis. The curves represent two experiments, one at each of two levels of CM. Cells were grown at 30°C in medium A'Ko' 3 supplemented with leucine and uracil. At a turbidity of 0·25 (zero time), portions were added to equal volumes of warm medium containing [14CJleucine, 0·014",c/ml., and [14CJuracil, 0·056 ",c/ml., with or without CM at the (final) concentrations indicated (in ",g/ml.). Incorporation of radioactivity into protein and into nucleic acid was followed by counting "hot" and "cold" TCA precipitates, as described by Dubin et al. (1963). Results are expressed as percentage of the control cultures. 0·25 ",g/ml. CM: - 0 - - 0 - , protein; - - • - - • - -, nucleic acid; 0·5 ",g/ml. CM: -l:,.--l:,. - , protein; - - A - - A - -, nucleic acid.

appeared contradictory, and suggested the need for more detailed studies on the effects of comparably low levels of CM. The usual concentrations of CM (approximately 20",g/ml.), like high levels of STM, affect both RNA and protein synthesis too rapidly to permit accurate timing. However, by using CM at concentrations of 0·25 to 0·5 ",g/m!., and incubating cultures at 30°C rather than 37°C, it was possible to detect a lag between the stimulation of RNA synthesis and the slowing of protein synthesis, as measured by incorporation of [14C]leucine. Figure 3 summarizes two such experiments. At 0·5 ",g/m!., the stimulation (measured by [14C]uracil incorporation) was near maximal by 10 minutes, a time when the rate of protein synthesis had just begun to decline measurably. At 0·25 p.g/ml., [14C]uracilincorporation was stimulated by 15% at four minutes, and by almost 40% at 30 minutes; even at this latter time,

STREPTOMYCIN AN.D RNA METABOLISM IN E. COLI

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protein synthesis was within 2 or 3% of normal. Cells such as those used for these experiments were found to contain one-third as much RNA as protein (approximately 150 p.g of RNA and 450 p.gof protein per mg of cells). Thus, on a weight basis, the stimulation of RNA synthesis by CM must have overbalanced the slight inhibition of protein synthesis, and the over-all rate of macromolecule synthesis must have been increased. Earlier studies on RNA and protein synthesis in CM-treated cultures of Aerobacter aeroqenes yielded similar results (Fraenkel, 1961), but thc precision of the data (obtained colorimetrically only) was thought inadequate to support definite conclusions. (c) Effects of streptomycin and chloramphenicol on M-RNA synthesis

The studies described in the previous sections might not have detected specific effects on M-RNA, which constitutes a small fraction of the total bacterial RNA (Levinthal, Keynan & Higa, 1962; Midgeley & McCarthy, 1962; Gros, Naono, Woese, Willson & Attardi, 1963). However, this fraction turns over so rapidly that 300r - - - - - - - - - - - - - - - - - - - - - , "Pulse"

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FIG. 4. Effects of streptomycin on "pulsed" and "net" RNA synthesis. Cells were grown as described for Fig. 1. At a turbidity of 0·2 (zero time), portions were added to equal volumes of warm medium containing sufficient [UC]guanine to give a specific activity of 0·1 fIoc/mg; one tube also contained STM, 60 p.g/m!. For following net RNA synthesis, 5- or 3-m!. samples were added to equal volumes of cold 10% TCA. For following "pulsed" R~A synthesis, 2-m!. samples were added to tubes containing (in 0·1 ml.) 0·04 flog of [14C]uracil, 91 floc/mg. The tubes were shaken manually for 30 sec, after which incorporation was stopped by rapidly adding 4 m!' of cold 7'5% TCA. The precipitates were processed as described in the text. The counts from the [14C]uracil were calculated by subtracting (from the total counts in each "pulsed" sample) the counts contributed by [UC]guanine, as estimated from the net uptake curves. Specific activities were adjusted to keep the total counts at least two times the counts from [14C]guanine. The concentration of uracil was adjusted so that the 30 sec point would be approximately midway along the steep portion of the uracil incorporation curve (cf. McCarthy & Britten, 1962).

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D . T. DUBIN

it can be preferentially labeled by exposing cells to a short pulse of radioactive precursor (Gros et al. , 1961). As reported for a similar system (Monier et al., 1962), the RNA labeled during a 30-second pulse of [l4C]uracil sedimentcd in the polydisperse fashion characteristic of M-RNA; therefore, pulses of this duration were used as a measure of M-RNA synthesis. Since ribosomal precursor RNA has also been reported to be significantly labeled in such a time period (Midgeley & McCarthy, 1962), the measure is admittedly a rough one; however, it should detect any gross discrepancies between effects on over-all (net) RNA synthesis and on M-RNA synthesis. Figure 4 summarizes an experiment in which the effects of STM on both net and "pulsed" RNA synthesis were studied. Net synthesis was followed by allowing cells to incorporate [l4C]guanine continuously, and pulsed synthesis by exposing small samples of the culture to [14C]uracil of much higher specific activity, as described in the legend. At a concentration of 60 ILg/mi. or STM, several points could be obtained during both the period of stimulation and the period of inhibition of RNA synthesis, before the background of accumulated [14C]guanine became too high. The results indicate roughly parallel effects on "pulsed" and net RNA synthesis (the latter as estimated from the slopes of the [14C]guanine curves). It should be noted that under the conditions of this experiment the STM-triggered depolymerization of RNA would not have progressed to the point where it might have complicated interpretation of these results. Comparable results were obtained for higher and lower concentrations of STM and for eM. (d) Molecular nature of the RNA accumulated by chloramphenicol- and streptomycin-treated cultures

The nature of the RNA accumulated by cultures exposed to various concentrations of STM or CM was studied by means of sucrose gradient centrifugation. RNA was labeled with radioactive uracil or guanine as in the studies on over-all RNA synthesis described in the earlier sections. Labeled cultures were kept small in volume (20 to 40 ml .), and were mixed at the time of harvesting with larger "carrier" cultures which had been exposed neither to a drug nor to a radioactive precursor. The carrier cultures contributed the bulk of the 260 mIL absorbancy to the final preparations, and provided markers for the normal RNA and ribosomal components of E. coli. In some experiments a "double-labeling" method was used. In this method, one of a pair of cultures was labeled with [14C]uracil and the other with [3H]uracil. The two cultures were combined at the time of harvesting, and the 14C and 3H of the final preparations were counted differentially as described in Materials and Methods. This method halved the manipulations required for processing a pair of cultures and also assured identical processing conditions for the pair. Figures 5 and 6 present results from an experiment in which this double-labeling technique was employed for three pairs of cultures. One culture (A) was labeled in the absence of drug, to serve as an added control. The other five were labeled under various conditions of drug treatment. Cultures Band C were treated with low concentrations of STM and CM, respectively, so as to obtain significant stimulation of RNA synthesis, with little or no inhibition of protein synthesis, during the 32-minute period of labeling. Cultures Dand E were treated with high levels of STM and C:~I, respectively, so as to assure virtual cessation of protein synthesis during the period

STREPTOMYCIN AND RNA METABOLISM IN E. COLI

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of labeling. Culture F, finally, was labeled only after 35 minutes of exposure to a low level of STM, so as to obtain a moderate inhibition of protein synthesis during the period of labeling. The cultures werelabeled with either [140] or [3H]uracil, and were harvested in pairs, as described in the legend to Fig. 5. I 0·4

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_FIG. 5. Sucrose gradient centrifugation patterns of crude extracts of cultures treated with streptomycin or chloramphenicol. A large.scale culture growing in medium A'K o' 3 was used as starting material. At a turbidity of 0·3 (zero time) three 400-mI. batches were poured onto crushed ice to provide carrier cells, At the same time 20·ml. portions were added to each of six tubes containing 10 ml. of warm medium plus ["C]- or [3H]uracil and/or a drug, at the foIlowing final concentrations. Culture A, [UC]uracil, 0·3I£c/mI.; no drug. Culture B, p'C]uracil, 0·3I£c/ml.; STM, 25I£g/ml. Culture C, [3H]uracil; CM, O·5I£g!ml. Culture D, [3H]uracil, 3I£c/ml.; STM, 10001£g/ml. Culture E, [3HJuracil, 3I£c/ml.; CM, 20 ~/ml. Culture F, no radioactive uracil, STM 25,..g/ml. At 32 min, cultures A and D were added to one batch of carrier cells, Band E to a second, and C to the third. At 35 min, ["C]uracil was added to culture F to give a concentration of 0·3I£c/ml.; 34 min later, this culture was also added to the third batch of carrier cells. Dialysed crude extracts were prepared and fractionated by centrifugation through a sucrose gradient (3'5 hr; 38,000 rev.rmin) as described in the text. Each fraction was assayed for 260 ml£ absorbancy (-0--0-), p'CJuracil (-e--e-), and [3HJuracil (-6-6-). Panel I corresponds to cultures A and D; Panel II to cultures B and E; and Panel III to cultures C and F.

Both crude cell extracts and purified RNA were prepared from these cultures. The crude extracts were centrifuged through sucrose gradients containing 1O-4 M -Mg2+. This procedure fractionated normal extracts into peaks corresponding to the 50 sand 30 s ribosomal subunits and a soluble (4 s) RNA fraction, as indicated by the 260 ml-'absorbancy patterns of Fig. 5. Centrifugal fractionation of the purified RNA preparations yielded 23 s, 16 sand 4 s peaks, as shown in Fig. 6. The patterns given by the control labeled culture (A) closely paralleled the corresponding 260 ml-'- absorbancy patterns (Panel I of Figs 5 and 6). This result indicates that the period of exposure to radioactive uracil was long enough to mask any preferential labeling of minor fractions such as M-RNA or ribosomal precursor RNA in the normally growing' culture.

D. T. DUBIN

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The patterns given by cultures Band 0 also paralleled their corresponding 260 mfL absorbancy patterns (Panels II and III of Figs 5 and 6). The higher ratios of labeled to carrier RNA in these cultures (relative to the control culture) reflect the stimulation of RNA synthesis. One can conclude that the low levels of both STM and OM stimulated the synthesis of each of the major stable fractions of E. coli RNA to comparable extents (as has been shown for low levels of OM (Kurland & Maalee, 1962)}; and that the ribosomal RNA was normally incorporated into 30 sand 50 s ribosomal subunits. 45

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FIG. 6. Sucrose gradient centrifugation patterns of the purified RNA from cultures treated with streptomycin or chloramphenicol. The cultures were those described in Fig. 5. RNA was prepared and fractionated by centrifugation through a sucrose gradient (8 hr; 35,000 rev. jmin), and fractions were collect ed and assayed as described in the text. The correspondence between patterns and cultures is as for Fig. 5. Absorbancyat 260 miL (-0-0-), [14C]uracil (-e -e-), [3H]uracil (-.6-.6-).

This latter conclusion was substantiated for low levels of STM by an experiment in which cells were labeled with both [140]uracil and [140]leucine. Crude extracts of these cells were fractionated in sucrose gradients and the fractions were assayed for newly-synthesized RNA and protein by counting " cold " and "hot" TOA precipitates (Dubin et al., 1963). The ratio of the [140)leucine to [14C]uracil in the 50 s subunits was found to be normal. (The 30 s subunits were not adequately separated from the soluble protein.) Hence, since there was no stimulation of over-all protein synthesis, there must have been some preferential synthesis of ribosomal protein in these cultures. A similar observation has been made for high levels of OM (Aronson & Spiegelman, 1961a).

STREPTOMYCIN AND RNA METABOLISM IN E. COLI

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The cultures treated with high levels of STM or CM (cultures D and E) yielded crude extracts the sucrose gradient patterns (Panels I and II of Fig. 5) of which differed markedly from the normal. Little radioactive material sedimented with the 30 sand 50s ribosomal subunits, whereas most sedimented with the soluble RNA or as a diffuse peak or shoulder which appears to correspond to the "15 s" CM particles described by Nomura & Watson (1959). The relative amounts of the 15 s and soluble material were somewhat variable from experiment to experiment, probably at least in part because of variable degrees of in vitro degradation of the 15 s material (see next section); in many experiments, the 15 s peak was as prominent as the 4 speak. Purified RNA from these cultures yielded sedimentation patterns (Panels I and II of Fig. 6) which were more nearly normal. However, there were slight increases in the amounts of 4 s RNA relative to R-RNA, as previously found for GM (Kurland & Maaloe, 1962); and the "16 s" peaks sedimented slightly faster than the normal 16 s RNA. This latter deviation was. quite reproducible. A similar shift in patterns observed by Okamoto, Sugino & Nomura (1962) and Nomura, Okamoto & Asano (1962) appears to have been disregarded. t Culture F was exposed to radioactive uracil during a period when one would have expected progressive slowing, and finally stopping, of protein synthesis due to the continuing action of a low level of STM. The sedimentation patterns for this culture (Panel III of Figs 5 and 6) were intermediate between those of the cultures in which protein synthesis had been normal or near-normal (A, B and C) and those of the cultures in which protein synthesis had been virtually nil (D and E). (e) Effects of streptomycin and chloramphenicol on the stability of RNA

Experiments were done to compare, in a general way, the stability of the RNA of cells treated on the one hand with STM, and on the other hand with CM:. Figure i summarizes a study on the stability of both newly-synthesized and pre-existing RNA in treated cells. RNA was labeled with [14C]uracil either before, or for 35 minutes after, addition of STM or CM; high and low levels of each drug were employed. After treatment, the cells were transferred to medium lacking drug but containing an energy source (glucose) and unlabeled uracil, and net RNA breakdown was followed by counting TCA precipitates of samples of culture taken at 3D-minute intervals. (This procedure would not have detected breakdown to TCA-precipitable polynucleotides. ) The effect of treating cells with a high concentration of CM was as previously described (Neidhardt & Gros, 1957; Horowitz, Lombard & Chargaff, 1958); namely, approximately one-half of the CM RNA (i.e., half of the newly-formed RNA) broke down, whereas the pre-labeled RNA was stable. These results support the inference that CM RNA is unstable because its R-RNA components lack normal association with ribosomal protein (Dagley, White, Wild & Sykes, 1962). The findings for STM were somewhat different, as one would predict from the earlier observation that this drug triggers an extensive breakdown of preformed RNA under the conditions of this experiment (Dubin & Davis, 1962). In STM-treated cells the breakdown of newly-formed RNA proceeded only slightly faster than that of the pre-labeled RNA, and the process did not level off as with CM. Furthermore, the low concentration of STM (which was, however, sufficient to cause extensive killing)

t After preparation of this manuscript, the paper of Mitsui, Ishihama & Osawa (1\)63) appeared, in which such a shift was noted for newly-synthesized "16 s" RNA.

760

D. T. DUBIN

triggered an even more rapid breakdown than did the high concentration. Possibly enough drug was carried over in the latter case to exert a transient stabilizing influence (see Feingold & Davis, 1962). 120r-------------------,

"0 ...

.....c: ou

'0

....c .,u ...

8c:

~

OIl

v

FIG. 7. The stability oj ENA in cells treated with streptomycin or chloramphenicol. Cells were grown, and exposed to drug, in medium A'Ko.s supplemented with uracil and leucine. At a turbidity of 0,1, the culture was divided into two portions, one of which (the "pre-labeled" subculture) received [14C]uracil, 0·027 Itc/ml. After growth for an additional 45 min, each culture was filtered through a 47-mm diameter HA Millipore filter and the cells were washed with, and then resuspended in, a small volume of fresh warm medium. Equal portions of the "pre-labeled" cells were then added to each of five tubes containing warm medium plus STM or CM at the concentrations indicated (ltg/ml.); one tube served as control. The other cells (the "post-labeled" cells) were similarly subdivided, except that the fresh medium contained [14C]uracil, 0·027 Itc/ml. At 35 min, each culture was filtered through a 24-mm diameter Millipore filter, and the cells were washed with medium A'K o.3 supplemented with non-radioactive uracil and leucine as before, but lacking (NH.).SO. and glucose. The cells were then resuspended (zero time) in medium of this composition, but containing glucose (0,2%). Samples were added to cold TCA, to be processed as described in the text, at 30-min intervals. The control cells (both pre-labeled and post-labeled) I08t approximately 5% of their counts in 2 hr. The acid-precipitable radioactive material of each treated culture is expressed as the percentage of the corresponding control. Open symbols represent pre-labeled, and solid symbols post-labeled, cells.

There are other striking differences between the OM- and the STM-triggered RNA breakdowns. First, the breakdown of OM RNA is inhibited by medium which allows cells to resume growth rapidly] (Neidhardt & Gros, 1957; Aronson & Spiegelman, 1961b); this is not the case for the STM-triggered breakdown, which of course takes place in cells which cannot resume growth by virtue of their having been killed by the antibiotic (Dubin & Davis, 1962). Secondly, the breakdown of preformed RNA in STM-treated cells requires a source of energy (Dubin & Davis, 1962), whereas the breakdown of OM RNA does not (Neidhardt & Gros, 1957). This second difference suggested experimental conditions under which one might test the stability of STM RNA in the absence of the superimposed generalized RNA

t For this reason, the cells in experiments such as that of Fig. 7 were resuspended in medium lacking S and a readily available source of N.

STREPTOMY CIN A N D RNA META BOLI SM I N E.

co t.t

761

brea kdown normally t riggered by STM. Cells were labeled with [14C]uracil either befor e, or during, exposure to a high level of STM, as in the experiment described in Fig. 7; RNA breakdown was followed , however , in a medium lacking glucose. The results resembled those presented in Fig. 7 for the high level of CM, in that only the newly-synthesized RNA (the STM RNA) broke down (see Table 1), and the process TABLE

1

Effects of glucose, 8treptomycin and chloramphenicol on the breakdown of RNA in cells treated with high levels of 8treptomycin or chloramphenicol

Besuspension medium

Complete'[ +STM +CM No glucose] +STM +CM

eM-treated cells STM-treated cells (eta/min at 2 hr, as (eta/min at 2 hr, as % of initial ) % of initial) Pre-labeled Post-labeled Pre-labeled Post-labeled 41 93 97

25 86 72

99

57 89 86

100

62 64 51

96

50 46 64

The experimental plan was essential1y as d escribed for Fi g. 7, except t hat, aft er t reat men t with STM or CM, the washed cells were resusp ended in media of varyi ng composition. Th e results are ex presse d as percentages of the ini t ia l radioacti ve material remaining a cid- p rec ip itable 2 hr after t his resu spension ; for untreat ed cultu res this figure ranged fr om 95 to 105 %. I n all cases STM was used at 1000 ~/ml . and CM at 20 ~/ml. t " Com plete" medium was medium A'Ko-s su p ple me n te d with gluc ose and n onradioa ctive uracil a nd leucine, but lacking (NH,l.SO" as de scr ib ed for Fi g. 7 ; "no glucose" m edium had the same composition ex cep t for the absence of glucose.

tended to level off after half of the radioa cti vity had been released. Hereafter, this limited, energy-independent RNA breakdown in STM-treated cells will be referred to as the " breakdown of STM RNA" and th e more exte nsi ve, energy- dependent br eakdown will be referred to as the "STM-triggered breakdown". Previous studies had shown t hat the STM-triggered breakdown of pr elabeled RNA is inhibited by high levels both of CM (Dubin & Davis, 1962) and of STM itself (Feingold & Davis, 1962), and that the breakdown of CM RNA is inhibited (in the presence of a readily available energy source) by CM (Neidhardt & Gros, 1957). Table 1 summarizes t he results of experiment s aimed at rounding out this picture. Cells were labeled with [14C]uracil either before or during exposure to a high level of STM or CM. The cells were then resuspended in various media and RNA breakdown was followed as before; the 120-minute points have been tabulated. Th e results show that (1) the STM-triggered breakdown of newly-synthesized RNA (like that of preformed RNA) is inhibited by both STM and CM; and (2) STM, like CM, can stabilize CM RNA, but (also like CM) t his stabilizing effect is energy depend ent. Sinc e it is not known to what extent the breakdown of STM RNA contributes to the STM-triggered breakdown of "post -labeled " RNA, one cannot state definitely whether, in the presence of glucose, STM and CM can also stabilize STM RNA. CM RNA is known to be relatively un stable in vitro (Nomura & Watson, 1959) as well as in vivo, and a similar in vitro instability was noted for STM RNA. When

762

D. T. DUBIN

RNA was prepared from crude cell extracts (such as those used to obtain the patterns of Fig. 5) rather than by treating cells directly with detergent and bentonite, patterns such as that in Fig. 8 were often obtained for both CM and STM RNA. Most of the R-RNA appeared to have been degraded to material sedimenting with, or only slightly ahead of, the 4 s RNA.

0·6

6

~ E o -o

~

0·4

>..

~

o

-D

o V)

-D

-c

20

30

Fraction no.

FIG. 8. Sucrose gradient centrifugation pattern of RNA purified from a crude extract of a streptomycin-treated culture. RNA was prepared as described in the text, except that a portion of a dialysed cell extract (the one used to obtain Panel I of Fig. 5) was used as the starting material rather than washed cells, and additional DNase treatment was omitted. Centrifugation and assay of fractions was essentially as described for Fig. 4. Since sedimentation was somewhat slower than for Fig. 6, only the top two-thirds of the pattern is shown. Absorbancy at 260 m,. (-0-0-), [UCJuracil (-e-e-), [3H]uracil (-6-6-).

4. Discussion The recent finding that low levels of STM stimulate RNA synthesis before detectable slowing of protein synthesis (Dubin et al., 1963) suggested that STM might have a direct effect on RNA synthesis. The present studies make this inter. pretation seem unlikely. The effects of low (as well as high) levels of STM have close counterparts in effects of comparable levels of eM on RNA and protein synthesis. There is good evidence that the protein-synthesizing system is the only site of direct action of eM (see review by Brock, 1961), and at least a major site for the direct action of STM (Flaks et al., 1962; Speyer et al., 1962; Davies, 1963). Hence it seems economical to view the stimulation of RNA synthesis by both drugs as secondary to more immediate effects on the bacterial protein-synthesizing system. However, how can a secondary effect precede a primary one? One can avoid this paradox by emphasizing the distinction between detectable slowing of protein synthesis and possible effects on the protein-synthesizing system. The following scheme utilizes this distinction in an attempt to explain the present results in terms of current ideas on the regulation of RNA synthesis in bacteria. In vitro studies with STM (Flaks et al., 1962; Speyer et al., 1962) and CM (Nathans & Lipmann, 1961) have indicated that each drug interferes with some (as yet undefined) step in the transfer of amino acids from charged S·RNA to protein. In cells treated with high levels of these drugs, such an action would have two prompt consequences, as suggested for CM by Kurland & Maalee (1962). Protein synthesis would stop and an accumulation of charged S·RNA (at the expense of the uncharged) would "derepress" RNA synthesis, as outlined earlier. It is proposed here that the ratio of

STREPTOMYCIN AND RNA METABOLISM IN E. COLI

763

charged to uncharged S-RNA is extremely sensitive to interference with the release of amino acids from charged S-RNA-so sensitive, in fact, that levels of STM or CM too low to produce detectable slowing of protein synthesis can still cause enough extra charging of S-RNA to increase this ratio significantly. In addition, the accumulation of charged S-RNA may itself exert a "homeostatic" influence in the presence of low levels of the drugs by partially counteracting any actual slowing of protein synthesis. This latter proposal finds support in studies on in vitro systems, in which an excess of "stripped" S-RNA (which presumably becomes charged in the system) has been shown to stimulate polypeptide synthesis in the presence of the usual inhibitory concentrations of STM or CM (Davies, personal communication). The above factors, either singly or in combination, could result in a steady state in which the high ratio of charged to uncharged S-RNA is maintained, the synthesis of all species of RNA is stimulated, and the synthesis of protein is apparently unaffected, Such a steady state' would be short-lived in the case of STM, which even at low concentrations causes progressive irreversible damage, probably involving the cell membrane as well as ribosomes (Dubin et al., 1963). However, with low levels of CM the steady state might be prolonged, producing the surprising phenomenon of stimulation of "growth" by a bacteriostatic agent. The slowing of RNA synthesis by leucine is a finding which bears further investigation. Possibly leucine, by repressing enzymes required for the synthesis of valine and isoleucine (Freundlich, Burns & Umbarger, 1963), caused a relative deficiency of these other amino acids. Thus leucine on the one hand, and STM and CM on the other, would have opposing effects on the degree of charging of S-RNA and hence on RNA synthesis. The suppression of the STM-induced stimulation of RNA synthesis by a complete mixture of amino acids appears to be the converse of the leucine effect. Presumably, in cells growing in the presence of an excess of amino acids, the level of uncharged S-RNA is so low that RNA synthesis is maximally "derepressed" even in the absence ofSTM. The experiments on the incorporation of [14C]uracil pulses indicate that the synthesis of M-RNA shares in the stimulation of over-all RNA synthesis by STM and CM. This result is in agreement with the inferences (1) that the stimulation is mediated by the degree of charging of S-RNA, (2) that S-RNA regulates RNA synthesis via its direct effects [Tissieres et al., 1963) on the activity of the DNAdependent RNA polymerase (Stevens, 1960; Weiss, 1960; Hurwitz, Furth, Anders, Ortiz & August, 1961), and (3) that all cellular RNA is synthesized by this enzyme (Hayashi & Spiegelman, 1961; Goodman & Rich, 1962; Yankofsky & Spiegelman, 1963). Agents such as STM and CM, if their action does in fact release inhibition of this enzyme, should be able to stimulate synthesis of all RNA types. As in earlier studies on CM (Kurland & Maalee, 1962), no disproportionate accumulation of M-RNA was observed. This may have been due to the fact that the centrifugal methods used are insensitive to changes in this minor fraction; in crude extracts, the M-RNA sediments with the 15 s particles, and in purified RNA preparations it sediments as a series of more or less ill-defined peaks, some of which overlap the R-RNA peaks. Base-ratio analyses, on the other hand, have indicated as much as a tenfold increase in the relative abundance of M-RNA after long exposure to CM (Midgeley & McCarthy, 1962); this tenfold excess, however, amounted to only about 10% of the OM RNA. The present results are compatible with such an

764

D. T. DUBIN

accumulation of M-RNA in either STM- or OM-treated cells; however, it is clear that the bulk of the newly-synthesized RNA in these cells is soluble or ribosomal RNA. The limited accumulation of M-RNA that was in fact detected in OM-treated cells was attributed to inhibition of the normal breakdown of this fraction (Midgeley & McOarthy, 1962). The other RNA-stabilizing actions of OM (see below) support such a hypothesis. However, a late preferential inhibition of R-RNA synthesis would (as described below for S-RNA) also contribute to any apparent excess of M-RNA. Several earlier studies employing centrifugal fractionation methods have suggested a more substantial accumulation in STM-treated cells of slowly-sedimenting RNA fractions (aside from normal S-RNA). Eaton & Oaffrey (1961) reported that STM caused the synthesis of an abnormal "soluble" RNA, which lacked amino acidacceptor activity. Two other groups have briefly noted the accumulation of a slowlysedimenting fraction which had "properties of" (Flaks et al., 1962) or "resembled" (Wolfe & Hahn, 1963) M-RNA. Since these authors do not appear to have taken special precautions against ribonuclease action, their results may be in part the consequence of in vitro degradation of the relatively labile RNA of the "15 s" particles. The finding that these abnormal 15 s particles accumulate in STM-treated cells subsequent to the overt slowing of protein synthesis is in general accord with previous findings in similar systems. Such particles were noted first in OM-treated cells (Dagley & Sykes, 1959; Nomura & Watson, 1959), subsequently in puromycintreated cells and in "relaxed" methionine auxotrophs starved of methionine (Dagley et al., 1962), and most recently in potassium-starved cells (H. Ennis & M. Lubin, personal communication)-four other cases of inhibition of protein synthesis with continuing RNA synthesis. Work with OM has further indicated that the RNA of these particles is R-RNA (Kurland, Nomura & Watson, 1962). The "STM particles" thus fit into an over-all pattern; the 15 s particles are apparently not specific for a particular drug, but are presumably ribosome precursors which accumulate when there is a deficiency of ribosomal protein (see Dagley et al., 1962). The slight increase in the sedimentation rates of the "16 s" RNA of the STM and the OM particles (compared with normal 16 s RNA) is at present unexplained. Possibly this RNA has a slightly higher molecular weight or more compact configuration before the 30 s ribosomal subunit is completed than afterwards. The increased accumulation ofS-RNA in cells the protein synthesis of which has been inhibited by STM is also in accord with studies on OM (Kurland & Maalee, 1962). The suggested interpretation might apply as well to STM: "protein is necessary for (ribosomal) RNA synthesis, and ... is consumed in the process. This protein must therefore be present in the normal cells in an amount corresponding to the quantity of ribosomal RNA synthesized at high OM concentrations." In the absence of new protein synthesis, lack of this protein would eventually limit the synthesis of R-RNA but not of S-RNA (or M-RNA). The similar stability properties of STM RNA and OM RNA support the view that the two are similar in molecular composition. In both cases, breakdown in vivo does not require energy, and involves only about half of the newly-synthesized RNA. In both cases, also, the R-RNA components (having been incorporated into 15 s particles) tend to break down in vitro to more slowly-sedimenting RNA species. It is likely (although by no means proved) that both the in vivo and the in vitro processes reflect the susceptibility of the RNA of the 15 s particles to enzymic

STREPTOMYCIN A N D RN A METABOLI SM IN E . GO L I

765

attack, and t hat the in vitro degradation corresponds to an early stage in the more complete in vivo process. The effects of STM in inhibiting t he breakdown of e M RNA, as well as in inhibiting t he STM.triggered breakdown, may well be du e to a dir ect intera cti on between t his polycationi c drug and RNA, such as has been demon strated (Cohen & Lichten stein, 1960). In the case of other polycati onic compounds, such an int era ct ion has been shown to prote ct RNA against enzymic degradation in vitro (see Tabor , Tabor & Ro senthal , 1961). Th e similar pro tective effects of CM: ar e less easily explained . A dir ect int eraction bet ween C~1 and CM RNA has been postulated (Dagley et al., 1962), a nd a recent report has actually demonstrated some affinity betw een CM and normal ribosomes (Vazquez , 1963). However, the evidence that eM can stabilize RNA dir ectl y must be considered meagre at this time. Even more than with STM, t herefore, one must entertain the possibility that stabilization of RNA by CM is secondary (in some unknown manner) to the primary action of the drug on t he protein-synthesizing system. The generalized breakdown of RNA in STM-treated cells (th e "STM-triggered breakdown") appears quite different from the breakdown of CM RNA or STM RNA. It involves preformed as well as newly- synthesized RNA; it occurs after treatment with either high or low levels of the drug; and it continues even afte r three-quarters of the to t al RNA has been degraded. Hence, it must involve the RNA of normally assembled ribosomes, as well as normal S·RNA. Although its nature is not understood, this process serv es as an example of the complex secondar y cha nges which can occur in cells killed by STM, as compared with cells merely inhibited by CM. It has been assumed for simplicity that effects on the net brea kdow n of RNA were, in fact, effects on RNA stab ility. H owever, neither this, nor earlier, work has ruled out the possibility that STM RNA and CJI,:I RNA norma lly undergo rapid met aboli c turnover; that the initial breakdown products normally enter a pool that is not completely accessible t o external precursors; and t hat STM and eM do not inhibit breakdown, but rather enh ance resyn thesis, of this RNA. t In t he absence of these drugs, this pool of br eakdown products might increase and serve as t he source of the large amounts of acid-soluble material which eventua lly accumulate . Th is hypothesis would explain the energy dep endence of the " protect ive" effects of eM (cf. Neidhardt & Gros , 1957) and of STM. However , t here are other possible explanations for this energy dep endence ; for example, upt ake or retention of the drugs may require energy. It is clear that further work will be needed to provide an adequate understanding of the breakdown of RNA in STM· and CM-treated cells. The present studies by no means explain the lethal action of STM, but do allow for some simplificat ion of the over-all picture. The early stimulation of RNA synthesis by low levels of t he drug can now be ascribed to an otherwise sub liminal effect on the protein-synthesizing system. In addition, t he accumulati on of slowly -sedimenting RNA fractions can be ascribed to a more ovett interferen ce with protein synthesis. These interpretations serve to emphasi ze t he importance of t he direct action of STM on ribosomes (Flaks et al. , 1962; Speye r et al., 1962), an action which can now be

t I sot opic stud ies have in fac t indica t ed some t u rnove r of CM RNA in the presen ce of eM (Neidha rd t & Gros, 1957; H oro witz et al., 1958), b ut t he m agnitude of t he t urnover was insu fficien t t o allow one to ascribe the stabilizing acti on of CM to effects on resynt hesis a lone.

766

D. T. DUBIN

considered to begin as early as the action of the drug on the cell membrane (Dubin et al., 1963). Recent work has excited renewed interest in possible close associations in bacteria between the cell membrane and macromolecule-synthesizing systems (Abrams & Nielsen, 1963; Suit, 1963); perhaps the two seemingly separate early actions of STM reflect an action on a single component which is necessary for the normal function of both these systems. I should like to thank Dr. Bernard Davis for his support and advice; Mrs. Ruth Darrow and Mrs. Avril Elkort for their expert technical assistance; Dr. Elmer Pfefferkorn for a generous gift of recrystallized sodium dodecyl sulfate; and Dr. Kimiko Asano for several very helpful discussions. This work was supported by grant no. G9078 to Dr. Bernard Davis from the National Science Foundation. REFERENCES Abrams, A. & Nielsen, L. (1963). Fed. Pmc. 22, 353. Ames, B. N. & Dubin, D. T. (1960). J. Biol. Chem, 235, 769. Anand, N. & Davis, B. D. (1960). Nature, 185, 22. Aronson, A. I. & Spiegelman, S. (1961a). Biochim. biophys. Acta, 53, 70. Aronson, A. I. & Spiegelman, S. (1961b). Biochim. biophys. Acta, 53, 84. Asano, K. (1963). Fed. Proc. 22, 524. Bray, G. A. (1960). Analyt. Biochem. I, 279. Britten, R. J. & Roberts, R. B. (1960). Science, 131, 32. Brock, T. D. (1961). Bact. Rev. 25, 32. Chen, P. S., Toribara, T. Y. & Warner, H. (1956). Analyt. Ohern, 28, 1786. Cohen, S. S. & Lichtenstein, J. (1960). J. Biol. Chern, 235, PC55. Crestfield, A. M., Smith, K. C. & Allen, F. W. (1955). J. Biol. Ohern: 216, 185. Dagley, S. & Sykes, J. (1959). Nature, 183, 1608. Dagley, S., White, A. E., Wild, D. G. & Sykes, J. (1962). Nature, 194, 25. Davies, J. (1963). Abstr, 145th Meeting Amer. Ohem; Soc., 14C. Dubin, D. T. & Davis, B. D. (1962). Biochim. biophys. Acta, 55, 793. Dubin, D. T., Hancock, R. & Davis, B. D. (1963). Biochim. biophys. Acta, 74, 476. Eaton, N. R. & Caffrey, R. (1961). J. Bact. 81, 918. Feingold, D. S. & Davis, B. D. (1962). Biochim. biophys. Acta, 55, 787. Flaks, J. G., Cox, E. C. & White, J. R. (1962). Biochem. Biophys. Res. Oomm. 7, 385. Fraenkel, D. G. (1961). Doctoral thesis, Harvard University. Fraenkel, D. G. & Neidhardt, F. C. (1961). Biochim. biophys. Acta, 53, 96. Fraenkel-Conrat, H., Singer, B. & Tsugita, A. (1961). Virology, 14, 54. Freundlich, M., Burns, R. U. & Umbarger, H. E. (1963). In Informational Macromolecules, ed. by H. J. Vogel, V. Bryson & J. V. Lampen, p. 287. New York: Academic Press. Gale, G. P. & Folkes, J. P. (1955). Biochem. J. 59, 675. Goodman, H. M. & Rich, A. (1962). Proc. Nat. Acad. Sci., Wash. 48, 2101. Gros, F., Hiatt, H., Gilbert, W., Kurland, C. G., Risebrough, R. W. & Watson, J. D. (1961). Nature, 190, 581. Gros, F., Naono, S., Woese, C., Willson, C. & Attardi, G. (1963). In Informational Macromolecules, ed, by H. J. Vogel, V. Bryson & J. V. Lampen, p. 387. New York: Academic Press. Hahn, F. E. & Ciak, J. (1959). Bact. Proc, 1959, p. 131. Hayashi, M. & Spiegelman, S. (1961). Proc. Nat. Acad. Sci., Wash. 47, 1564. Horowitz, J., Lombard, A. & Chargaff, E. (1958). J. Biol. Ohem, 233, 1517. Hurwitz, J., Furth, J. J., Anders, M., Ortiz, P. J. & August, J. T. (1961). Oold Spr, Harb. Symp. Quant. Biol. 26, 91. King, E. J. (1932). Biochem. J. 26, 292. Kurland, C. G. (1960). J. Mol. Biol. 2, 83. Kurland, C. G., & Maaloe, O. (1962). J. Mol. Biol. 4, 193. Kurland, C. G., Nomura, M. & Watson, J. D. (1962). J. Mol. Biol. 4, 388.

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50