The synthesis of messenger RNA without protein synthesis

J. Jlfol. Bioi. (1964) 8; 629-637

The Synthesis of Messenger RNA without Protein Synthesis I. Studies with Thymineless Strains of Escherichia coli J. L. STERN, M. SEKIGUCHI, H. D. BARNER AND S. S. COHEN Department of Biochemistry, Ul1iversity of Pennsylvania School of Medicine Philadelphia, Pennsylvania, U.S.A. (Received 9 December 1963) Escherichia coli, deficient in the ability to synthesize thymine, uracil and an amino acid, synthesize a small fraction of their normal RNA from uracil in the absence of thymine and the amino acid. The synthesis of even this fraction of RNA is inhibited by concomitant DNA synthesis. Most (75%) of the RNA made appears on the ribosomes and can be degraded on these structures in the presence of inorganic phosphate. When synthesized in the presence of an inducer of p-galactosidase, the RNA appears to permit the rapid synthesis of a small amount of this enzyme in the apparent absence of the inducer. These observations are consistent with the hypothesis that the RNA made in the absence of an essential amino acid and of protein synthesis is largely messenger RNA.

1. Introduction We have been studying strains of Escherichia coli which are deficient in the ability to synthesize thymine, uracil and an amino acid (Kanazir, Barner, Flaks & Cohen, 1959), in order to explore the possibilities of separating the biosyntheses of DNA, RNA and protein, and to determine the biological consequences of non-integrated biosyntheses. The incubation of random cultures of such strains with thymine alone has been the basis of a method of synchronization (Maalee & Hanawalt, 1961) and we have been particularly interested in studying the properties of cells treated in this way. In the course of one such investigation we compared nucleic acid synthesis in deficient cultures, and observed a slight but significant incorporation of [2. 14C]uracil into RNA in the absence of thymine and an essential amino acid. This incorporation amounted to about 10% of that in the presence of the amino acid. Although incorporation of components into RNA in the absence of protein synthesis had been observed in the past, these observations had not been thought to be significant, or, if shown to be real and reproducible, even if slight, had been virtually ignored (Goldstein & Brown, 1960). In one important instance, the phenomenon of RNA synthesis without protein synthesis arose from the loss of a genetically determined control of the synthesis of ribosomal RNA (Borek & Ryan, 1958; Stent & Brenner, 196]) and was not revealed by a marked diminution of RNA synthesis. Indeed, this phenomenon shown by "relaxed" strains does not appear to be related to the observations which we are reporting. In our systems the low incorporation of uracil into RNA was markedly inhibited in the presence of thymine, leading us to suppose that the observed RNA synthesis might be that of messenger RNA which was affected by competition between synthesis of DNA and this class of RNA for a common template, i.e. DNA, at the bacterial chromosome. In this first paper we present incorporation data bearing on 629

630

J. L. STERN, M. SEKIGUCHl, H. D. BARNER AND S. S. COHEK

this hypothesis obtained with these thymineless polyauxotrophs in a number of different conditions of growth. However, the difficulties of proof of this hypothesis with " normal" cells have led us to carry out more rigorous tests with phage-infected bacteria, the results of which are presented in the following paper (Sekiguchi & Cohen, 1964). In that paper it is show n that messenger RNA can indeed be synthesized 'without protein synthesis in phage-infected bacteria, as is now proposed on the basis of the experiments presented in this communication.

2. Materials and Methods (a) Ohemicals [2· 14C]uracil was obtained from the California Corporation for Bio ch emical Research. TMGt was purchased from Mann Research Laboratories. Chloramphenicol was a gift from Eli Lilly and Company, Indianapolis, Ind., U.S.A.

(b) Bacteria

Two polyauxotrophic mutants of E. coli strain 15 were used in these experiments. Strain TAU, which requires thymine, arginine and uracil for its growth, has been described in earlier papers from this laboratory (Barner & Cohen, 1957; Kanazir et al., 1959). Strain THU is a newly-isolated mutant which is sensitive to many phages and requires thymine, histidine and uracil. E. coli strain THU was isolated from colicine-resistant (OR) strain of 15H- T-, which was furnished by Dr. T. Okada of Kanazawa University, Japan. Selection of the uracildeficient st r ain was effected after four cycl es of u.v. irradiation followed by penicillin screening according to the procedure of Lubin (1962). From previous records (Ryan, Fried & Mukai, 1955; Okada, Yanagisawa & Ryan, 1960) the pedigree of these mutants may be summarized as follows: 15--+ 15H---+ 15H-OR--+ 15H- T-OR--+ 15H- T- V-OR (THU). Nutritional tests have revealed that the combination of thymine, histidine and uracil is essential for the growth and multiplication ofTHU. Uracil can be replaced by cystosine. Although it has been reported that in some histidine-requiring mutants histidine can be replaced by adenine (Luzzati & Guthrie, 1955; Shedlovsky & Magasanik, 1962), such replacement will not permit growth of strain THU. However, the addition of adenine to a medium containing histidine slightly stimulates the rate of growth of THU. (c) Growth of bacteria

Bacteria were grown in a synthetic medium containing per liter : 16·5 g Na tHP0 4; 1'5g KH 2P04; 2'Og (NH4)2S04; 200mg MgS0 4,7H tO; 10mg CaCl t ; 0'5mg FeS0 4,7H tO and I g glucose. For the growth of THU this medium was supplemented with 20/Lg L-histidine, 10 /Lg uracil and 2 /Lg thymine/mi. In the case of TAU, L-arginine (20/Lg/mi.) was added in place of histidine. The growth rates of THU and TAU in these media are lIO and 60 min respectively per doubling of cell mass, as measured turbidimetrically. The cells were cultivated by incubating at 37°C with vigorous aeration, harvested at a concentration of 2 x 108 cells/ml., and washed once in the mineral medium. (d) Measurement of incorporation of [140]urcreil

Portions of the bacterial suspension were mixed with an equal volume of 10% trichloroacetic acid in an ice bath. One portion of the mixture containing 2 X 10 8 cells was filtered by means of Millipore filters (pore size 0·45 /L; diameter 24 mm), and washed with cold 5% TCA and distilled water. Another portion of the mixture containing the same amount of cells was incubated with 0·3 N-KOH at 37°C for 20 hr and then acidified to a final concentration of 0·5 N·TCA. After standing for 30 min in th e cold , the precipitate formed was collected on a Millipore filter and washed as described above. Although this procedure was successfully carried out with strain THU to separate DNA and RNA, the

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RNA SYNTHESIS IN THY:\UNELESS STRAINS

631

precipitation of DNA from the alkali- treated sample of strain TAU is incomplete. In this case 3 vol. ethanol were added to the acidified solution of alkali-treated material to give complete precipitation. The filter was pasted to a planchet and the radioactivity was measured directly in a gas-flow counter. The radioactivity of the untreated sample gives the amount of isotope incorporated into both nucleic acids, and the value for the alkali-treated sample gives that into DNA alone; the incorporation of isotope into RNA was calculated by subtracting the activity in DNA from that of the untreated sample. A multiplication factor of 1·25 was used to convert the incorporated radioactivity into micromoles. This factor was determined by growing cells in a medium complete except for a defined limiting amount of radioactive compound. When growth ceased and all the isotope had been incorporated, samples were taken in the manner described and the true values were compared to those determined experimentally. A 20 to 25% decrease due to self-absorption was obtained with 2 x lOs cells. Larger numbers of cells increased the self-absorption. (e) Preparations of ribosomes and RNA

Ribosomes were extracted from cells ground with alumina by 0·05 M-tris, pH 7·5, 0·005 M.MgClz and purified by differential centrifugation as described in a previous paper (Sekiguchi & Cohen, 1963). RNA was isolated from the ribosomal fraction by the Duponolphenol method (Gierer & Schramm, 1956).

3. Results (a) RNA synthesis in the absence of the amino acid When E. coli strain TAU, harvested during the exponential phase of growth, is incubated with arginine and [14C]uracil, a rapid incorporation of uracil into an acidinsoluble fraction (RNA) is observed. This occurs for a period of about 50 minutes at a rate comparable to that in multiplying cells, as can be seen in Fig. 1. In media 7 N

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FIG. 1. Incorporation of [2. 14C]uracil in E. coli strain TAU in both (a) random and (b] synchronized cells. Random cells are those harvested in exponential growth. Synchronized cells have been previously exposed to a 90-min incubation period in mineral media plus glucose and thymine. At zero time the concentration of cells was 2 X IOs/m!. and the concentration of cells in each sample was maintained at this level. [14C]U = [2. 14C]uracil; T = thymine; A = arginine; -e-e-,[UC]U+T+A; - 0 - 0 - , [14C]U+A; -.6-.6-, [14C]U;-X-x-, [14C]U+T.

632

J. L. STERN, 1\1. SEKIGUCHI, H. D. BARNER AND S. S. COHEN

lacking arginine, however, the initial rate of uracil incorporation is about 10% of that in the presence of the amino acid. Significant inhibition of even this low level of incorporation is effected by adding thymine to the medium. The quantitative and qualitative significance of the latter point was underlined by the experiment with synchronized cells presented in Fig. 1, in which the presence of thymine did not affect uracil incorporation. In cells which are synchronized by preincubation for 90 minutes in thymine alone, as in this experiment, DNA synthesis cannot be initiated unless the cells are given uracil and arginine, as well as thymine.

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FIG. 2. Interactions of pyrimidine incorporation in the absence of protein synthesis in random cells of E. coli strain TAU. The calculated value for RNA was obtained by assuming the thymine incorporation to be equal to that of the uracil in the DNA, and subtracting this value from the total nucleic acid incorporation. [14CJU = [2. HC]uracil; T = thymine; [UCJU; - x - x - , [14C]U+T; - 0 - 0 - , U + [14CJT; -D-D-, [HCJT; - 0 - 0 - , RNA calculated.

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Using isotopic uracil of higher specific radioactivity, we have studied in greater detail the incorporation of uracil in the absence of the amino acid. Typical results are presented in Fig. 2, as well as those on the incorporation of [2_ 14C]thymine into DNA in the presence and absence of uracil. In the latter case, the presence of uracil does not depress incorporation of thymine, and it appears that in the absence of exogenous uracil there must be an adequate endogenous source of uracil to supply the cytosine for DNA synthesis. Actually, the endogenous supply of uracil via RNA turnover in strain TAU had been discovered earlier in this laboratory (Kanazir et al., 1959). From Fig. 2 it can be seen that after correcting the uracil incorporation in the presence of thymine for that presumably used for cytosine in DNA, the presence of thymine inhibits RNA synthesis in TAU by about two-thirds. As can be seen in

633

RNA SYNTHESIS IN THYMINELESS STRAINS

Fig. 3, this degree of inhibition in TAU could not be increased by adding other deoxyribosides to the medium; hence thymine is truly limiting in DNA synthesis in this stram, To confirm this point, uracil incorporation into the separate nucleic acids, RNA and DNA, has been studied in strains TAU and THU; the results are shown in Fig. 4. The extensive inhibition of RNA synthesis by thymine has been confirmed for both strains. Furthermore, the inhibition detectable in THU is even greater than that

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FIG. 3. Effect of deoxyribonucleosides and of thymine on [2- 14C]uracil incorporation in random cells of E. coli strain TAU. ADR = deoxyadenosine; GDR = deoxyguanosine; TDR = thymidine; [14C]U; - 0 - 0 - , [14C]U+ADR+GDR; - X - x - , [14C]U+T; -6.-6.-, p'C]U +TDR; - 0 - 0 - , [14CJU+TDR+ADR+GDR.

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FIG. 4. Effect of thymine on the incorporation of uracil into the nucleic acids of random E. coli strains (a) TAU and (b) THU, in the absence of protein synthesis. [14CJU = [2. 14C]uracil; '1' = thymine; - 0 - 0 - , [14C]U RNA; [14CJU DNA; - - X - - x - -, [UC]U +'1' RNA; --6.--6.--, [14C]U+TDNA.

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634

J. L. STERN, M. SEKIGUCHI, H. D. BARNER AND S. S. COHEN

in TAU, since the presence of thymine has produced an absolute cessation of RNA synthesis for a short interval concomitant with a burst of DNA synthesis. In these systems, therefore, there appears to be a competitive relation between DNA synthesis and that portion of RNA synthesis which can occur in the absence of amino acids. Isolation of the ribosomal fraction of TAU revealed that the uracil incorporated was present mainly (about 75%) in this fraction. Such RNA labeled by [14C]uracil incorporation was labile and became selectively degraded to an acid-soluble form (30% in an hour) by incubation of the purified ribosomes in the presence of inorganic phosphate and Mg2+ (Sekiguchi & Cohen, 1963). The nature of the labeled RNA in this fraction has been studied more completely with strain THU. At this stage of the analysis the results were consistent with the possibility that the RNA synthesized in the absence of DNA and protein synthesis was messenger RNA, which was then released from the chromosome and transferred to the ribosomes. (b) Ohloramphenicol-stimulated RNA

As can be seen from Fig. 5, the presence of chloramphenicol produces at least a fourfold stimulation of RNA synthesis in the absence of amino acids in this system. Some additional experiments were done to determine if the chloramphenicolstimulated RNA was similar to the small amount of RNA made in the absence of 7-

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FIG. 5. Effect of chloramphenicol on uracil incorporation in (a) random and (b) synchronous cultures of E. coli strain TAU in the absence of DNA and protein synthesis. The chloramphenicol concentration was 20,..gjml. [14C]U = [2. 14C]uracil; A = arginine; CM = chloramphenicol; - 0 - 0 - , [14C]U+A;- x - x - , [14C]U+A+CM; [14C]U+ CM; -6 -6 -, [14C]U.

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amino acid. As can be seen from Fig. 6, the addition of thymine to the chloramphenicol-stimulated random culture did not reduce the initial rate of uracil incorporation by significantly more than the thymine inhibition of the uracil incorporation in the absence of the antibiotic. In synchronized cells which are stimulated similarly by chloramphenicol, thymine did not inhibit the stimulated rate. The

635

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lack of inhibition by thymine of the fraction of RNA synthesized in the presence of chloramphenicol suggests that chloramphenicol-stimulated RNA is not messenger RNA. In other systems also, the nature of the RNA made in chloramphenicol has not yet been completely clarified.

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FIG. 6. Effect of thymine on the chloramphenicol stimulation of RNA synthesis in random and synchronous cultures of E. coli strain TAU, in the absence of DNA and protein synthesis. p'C]U = [2. uC]uracil; T = thymine; CM = chloramphenicol; - 0 - 0 - , [UC]U; - x - x-, [uC]U +T; -6.-6.-, [UC]U +CM; - 0 - 0 - , [UC]U +T+CM.

(c) Synthesis of f3-galactosidase in strain THU

Since it appeared possible to charge ribosomes with a labile RNA presumed to be of the messenger variety, it was asked if it could be shown that such RNA made in the presence of an inducer could be used in the synthesis of an inducible enzyme. Strain THU was used for such studies on the induced biosynthesis of f3-galactosidase. The organism was starved for uracil and histidine and incubated for 10 minutes in a medium containing uracil, a mixture of amino acids lacking histidine, and an inducer. Glucose was replaced by succinate to minimize repression by catabolites (Nakada & Magasanik, 1962). Strain THU was grown aerobically at 37°C in medium containing inorganic salts, amino acids, adenine, thymine and uracil, with succinate as carbon source. The cells were harvested in the exponential phase of growth, resuspended in warm medium containing no histidine and incubated for 10 minutes in this "starvation" medium. The culture was divided into three parts and supplemented as follows: (1) uracil, 20p.g per ml.: (2) uracil and TMG, 0·1 mg per ml.: (3) uracil, TMG and thymine, 10 fLg per rnl, Incubation was continued for 10 minutes in these media. Each culture was chilled, sedimented and the cells washed. They were resuspended at 2 x 109 per ml. in warm medium containing inorganic salts, adenine, succinate and amino acids including histidine. Uracil was omitted from all media. The only variable was the content of TMG. Lrnl. samples were removed over a 1O-minute period of incubation in the synthesis media and assayed for f3-galactosidase activity (Hestrin, Feingold & Schramm, 1955).

636 .T. L. STERN, M. SEKIGUCHI, H. D. BARNER AND S. S . COHEK

Five such experiments were done. In each instance, of which a typical experiment is presented in Fig. 7, cultures preincubated in uracil and TMG rapidly developed a short burst of enzyme synthesis on supply of histidine in the absence of TMG. The development of this capacity was not inhibited by the presence of thymine. Cultures preincubated with uracil but without TMG did not increase their enzyme content on addition of histidine.

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FIG. 7. II·Galactosidase activity in strain THU preincubated with uracil and TMG, and then supplemented with histidine. The preincubated cells were resuspended in media containing amino acids including histidine, adenine. succinate and (a) no further additions; or (h) 0·1 mg /ml. TMG. Preincubation m edium is indicated as follows: uracil; - X - X - , uracil+TMG ; - 6 - 6-, uracil+TMG+thymine. The enzyme unit is defined as that amount of enzyme which will hydrolyse 0·01 ,.mole of substrate/min under the following conditions: 0·001 MoO.nitrophenol. Il·D.galactopyranoside, 0·14 M·sodium phosphate, pH 7·2 at 37 °C.

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4. Discussion We have shown that a small fraction of the total RNA is synthesized in the absence of protein, and that it is transferred to ribosomes where it can be selectively degraded. The inhibition of its synthesis under conditions of a concomitant DNA synthesis is also clear, leading to the idea that the RNA made may be produced on the bacterial DNA. We have also obtained evidence to suggest that such RNA made in the presence of the inducer, TMG, can be used in the production of ,a-galactosidase, ostensibly in the absence of an inducer. These points are at least consistent with the concept that the RNA so made is messenger RNA. To develop the evidence more convincingly we have turned to phage-infected bacteria, in which the unique properties of phage-induced messenger RNA are more clearly demonstrable. Such properties are base composition, metabolic lability, electrophoretic mobility and function in the production of phage-induced proteins. We shall hold the discussion in abeyance until those results are presented in the following paper (Sekiguchi & Cohen, 1964).

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This research was supp orted b y Grant no. E3963 from t he Nat ion al Institu t e of Arthri ti s and Metabolic Diseases of t he Unit ed States Public H eal th Se rv ice. One of t he au thors (M . S.) holds a 1961 Fulbrigh t Travel Gr ant find is on leave fro m t he Departmen t of B ioch em istry, Kanazawa Univ ers it y Med ical Sc hoo l, K anazawa , Japan . REFERENCE S Barner , H. D. & Cohen, S. S. (1957 ). J. Bact. 74, 350. Borek , E . & R yan, A. (1958). J . Bact. 75 , 72. Gierer, A . .& Schramm, G. S. (1956) . Natur e, 177, 702. Goldstein, A. & Brown, E. J. (1960) . B i ochim. bioph ys. A cta, 44 , 491. H eetrin, S., F eingold, D. S. & Schramm, M. (1955). In Me thods in E nzymology, ed. by S. Colowick & N . Kaplan, vol. I , p . 241. N ew York: Academic Press . Kanazir, D., Barner, H. D., Flaks, J. G. & Cohen , S. S . (1959 ). Biochim, biophys. Acta, 34 ,341. Lubin, M. (1962). J. Bact. 83 , 696. Luezati, D. & Guthrie, R. (1955). J . B iol . Chern, 216, 1. Maaloe, O. & Hanawalt, P. C. (1961) . J . Mol. ei« . 3, 144. Nakada, D. & Magasanik, B. (1962). Biochim. biophys. Acta, 61, 835. Okada, T., Yanagisawa, K. & Ryan, F . J . (1960). Nature, 188, 340 . Ryan, F. J., Fried, P. & Mukai, F. H. (1955). Biochim. biophys. A cta, 18, 131. Sekiguehi, M. & Cohen, S. S. (1963) . J . eu« Chem , 238, 367. Sek ig uch i, M. & Cohen, S. S. (1964). J . Mol . B iol. 8, 638. Shedlovsky, A. E. & Magasanik, B . (1962). J. B iol. Chern, 237, 372 5. Stent , G. S . & Brenner, S . (1961) . Proc. N at. Acad. Sci., Wash. 47 , 2005.