- Email: [email protected]
Synthesis and turnover of phage messenger RNA in E. coli infected with bacteriophage T4 in the presence of chloromycetin
J. M ol. Biol. (1962) 5, 527-534
Synthesis and Turnover of Phage Messenger RNA in E. Coli Infected with Bacteriophage T4 in the Presence of Chloromycetin K OJI OKAMOTO ,
Y.
S u aINO AND M ASAYA SU NO:MURA
Lnstituie fo r P rotein R esearch, Osaka University, Osaka, Japan (R eceived 13 August 1962) Treatment of E. coli with ch loro m ycet in prior to infection with T4 allows both ribosomal RNA and soluble RNA to be synthesized after infection. Both base composition analysis and hybridization experiments with DNA showed that the majority, if not all, of the pulse-labelled RNA synthesized in CM-pretreated, T4-infected cells was phage messenger RNA. The synthesis of host messenger RNA is inhibited by phage under suc h conditions, as is the synt h esis of host DNA. Phage messen ger RNA synthes ized in CM-pretreated , T4.infeeted cells was shown to be d egraded and repolymerized into ribosomal RNA and soluble RNA in the presence of CM.
1. Introduction Although after T-even phage infection the synthesis of typical ribosomal and soluble RNA st ops , a small am ount of phage-specific RNA is synthesized (Volkin & Astrachan, 1956 ; Nomura, H all & Spiegelman, 1960; Brenner, J acob & Meselson, 1961). It is suggested that this "messenger" RNA carries the information for the proteins synthesized after ph age infection. A similar kind of RNA is also found in uninfected cells (Gros ~ al., 1961 ; Hayashi &Spiegelman, 1961). Mter phage infection very little host messenger RNA is synthesized and the inhibition of host protein synthesis by ph age seems t o be du e to the inhibition of host messenger RNA synthesis. In a previous paper (Nomura, Okamoto & Asan o, 1962) we have shown that treatment of E. coli with ohloromycetirr] prior to infection with T4 allows both ribosomal RNA and soluble RNA to be synthesized after infection. It was suggested that, upon infection, the phage DNA induces the synthesis of some specific protein which causes the inhibition of the synthesis of host ribosomal and soluble RNA. lithe inhibition of the synthesis of host messenger RNA by phage is due to the same mechanism as that of ribosomal and soluble RNA, it would be expected that the addition of CM before infe ction would also prevent the inhibition of host messenger RNA synt hesis. The experimental results pre sented in this paper show that, on the contra ry, this inhibition still occurs in the presence of CM. The majority, if not all , of the messenger RNA synthesized in CM-pretreated, T4-infected cells is phage messenger RNA. It thus appears that phage infection can cause the inhibition of host messenger RNA syn t hesis without first producing a protein. As a consequ ence of this study, turnover of phage messenger RNA in infe cted cells in the presence of CM was presumed and , in fact, demonstrated.
t Abbreviation used:
chloromycetin =
eM. 527
.'i28
K. OKAMOTO, Y. Sl:GIKO AXD
~1.
XO~lURA
2. Materials and Methods E. coli strain B growing exponentially in tris-glueose-Casamino acids medium was infected with bacteriophage T4r+ in the presence of tryptophan (50fkg/ml.) at a multiplicity between 10 and 20, as described in a previous paper (Nomura, Okamoto & Asano, 1962). E. coli strain B6 (an auxotrophic mutant requiring both arginine and uracil) was used instead ofB when [3H]nridine was used to label RNA. Excess uracil (30fkg/ml.) was added to tris-glueose-Casamino acids medium to grow cells. Exponentially growing cells were collected by centrifugation at room temperature, washed with tris-glucose-Casamino acids medium (without uracil), and resuspended in the same medium (without uracil). Cells were infected with T4r+ as described above. R~A was isolated directly from cells without a prior grinding with alumina. The experiment was terminated by the addition of azide (10- 2 :'<1) and the culture was immediately poured onto crushed ice. Usually an appropriate amount of carrier uninfeeted cells (about 1011 cells) was added. The cells were then centrifuged and the pellets, without washing, were suspended in the cold in 3 m!. of 10- 2 ::II:-tris, [) x 10- 3 ::II:-Mgh, containing 10- 2 M-azide. Both D'Nase (20 fkg/ml.) and lysozyme (200 fkg/ml.) were added and the solution frozen in a dry-ice bath. After 3 freezings and thawings, the pH of the solution was brought to 5·2 by the addition of 5 drops of 0·02 M-CH 3COOH. The solution was then treated with sodium dodecylsulphate (recrystallized material, final concentration 0·3%) at 37°C for 3 min, and then shaken at 37°C for 5 min with an equal volume of phenol saturated with 0·05 ::II:-aeetate (pH 5,2)-0,01 M-Mg2+ solution. After centrifugation, RNA was precipitated with 2 vo!. alcohol, dissolved in 0·02 M-aeetate (pH 5,2) containing 0·02 M-KCI and 0·01 M-:\fg2+, and repreeipitated with alcohol. The final precipitate was dissolved in the same aeetate-KCI-MgCI 2 solution and dialysed overnight against 200 vol. of the same solution in the cold, with several changes of outer solution. With the present technique we consistently obtained phage messenger RNA with a sedimentation coefficient considerably higher than the value of 8 s reported in earlier papers (~omura et al., 1960; Gros et al., 1961). An example of a sedimentation pattern of phage messenger RNA obtained by this technique is shown in Fig. 2(a). This result is in accord with the recent results obtained in several other laboratories (Otaka, Mitsui & Osawa, 1962; Monier, Naono, Hayes, Hayes & Gros, 1962; Sagik, Green, Hayashi & Spiegelman, 1962). Hybrid formation of messenger RXA with heat-denatured DNA and the equilibrium density gradient centrifugation in CsCI were performed in a manner similar to that described by Hall & Spiegelman (1961). Heat-denatured D~A was prepared by heating a solution of DNA at 100°C for 15 min and then cooling rapidly in an icc bath. A UP-labelled RKA preparation was mixed with 10 fkg of heat-denatured DKA in 0·5 ml. of 0·3 M-~aCI 0·03 M-sodium citrate solution and incubated at 40°C for 36 hr. Ten fkg of heat-denatured DNA was then added and the mixture made up to 4 ml. with CsCI solution, such that the final concentration of CsCI was 57% (w/w). The solutions were centrifuged at 33,000 rev./min near O°C for 60 hr in a swinging bucket rotor, SW39, in a Spinco model L ultracentrifuge. At the end of the run, the tube was pierced with a needle and fractions of 10 drops each were collected. After suitable dilution of the fractions, ultraviolet absorption at 260 mfk and acid precipitable S2p of each fraction were determined. Phage 1'4 DNA was prepared according to Mandell & Hershey (1960). E. coli DNA was prepared by the method of Marmur (1961). E. coli "messenger RNA" used as a reference standard in the experiment of Fig. 1 was prepared from a "step-down culture" (Hayashi & Spiegelman, 1961). Cells were grown in modified Pcnassey-broth (Nomura et al., 1960) to a titre of 3 x 108/ml., harvested, washed and suspended in the tris-glueose synthetic medium (without Casamino acids). [32P]orthophosphate was then added for 5 min at 37°C. Cells were quickly chilled and RNA was isolated as described above. Sedimentation analyses of 3H-labelled RNA in sucrose density gradient with 32P-labelled RKA as sedimentation markers are performed as described in the following paper (Nomura, Matsubara, Okamoto & Fujimura, 1962). The analysis of RNA base composition was described in previous papers (Nomura et al., 1960; Xomura, Okamoto & Asano, 1962).
T4 MESSENGER RNA AFTER CHLOROMYCETIN TREATM:ENT
529
3. Results (a) Nature of messenger RNA synthesized in cells treated with OM before infection
In our previous paper, characterization of ribosomal and soluble RNA synthesized in eM-pretreated infected cells was described (Nomura, Okamoto & Asano, 1962). In order to characterize the messenger RNA, two kinds of techniques were used. One involves the formation of specific DNA-RNA hybrids. The other is the determination of the base composition of the [32P]RNA synthesized after a short exposure to [32P]orthophosphate at low temperature. Under such conditions, the newly synthesized [32P]RNA is mostly messenger RNA (Gros et al., 1961). TABLE
1
Base composition of 32 P pulse-labelled RNA synthesized in T4-infected cells treated with chloromycetin (OM) before infection s2P-pulse
o sec
CM: (before infection)
at 25°C
10 min at 37°Ct phage DNAt E. coli DNA§ E. coli ribosomal RNA'\[
+ + +
Base composition (A = 1,00)
Phage C
A
G
U
+ +
0·60 0·59 0·96
1·00 1·00 1·00
0·81 0·82 1·25
1·10 H2 1·15
+
0·85
1·00
1·27
0·83
0·53 1·09 0·88
1·00 1·00 1·00
0·56 1·14 1·26
1·0011 1·0811 0·86
Culture of E. coli B exponentially growing in tris-glucose-Casamino acids medium at 37°C (2 x lOs/mI.) was transferred to a 25°C bath and incubated for 10 min. The culture was then treated with CM: (100 f.Lg/mI.) and, 6 min later, infected with T4 at a multiplicity of 10 in the presence of tryptophan (50 f.Lg/mI.). [32P]orthophosphate was added 5 min after infection. Assimilation of 32p was stopped 40 sec later. Control cultures without either CM: or phage infection were also analysed.
t Data published in a previous paper (Nomura, Okamoto & Asano, 1962). t Wyatt (1953). § Dunn & Smith (1958). 'II Spahr & Tissieres (1959). II Value for thymidylic acid instead of uridylic acid. First, experiments were performed at 37°C as previously described (Nomura, Okamoto & Asano, 1962). CM (80 JLg/ml.) was added 6 minutes before infection and [32P]orthophosphate was supplied between 4 and 14 minutes after infection. The entire culture was then chilled and RNA was prepared. This preparation was shown to contain both ribosomal RNA (16 sand 23 s) and soluble RNA (4 s) as the major 32P-Iabelled components. This preparation was subjected to the hybridization process with heat-denatured T4 DNA and with heat-denatured E. coli DNA. With heatdenatured phage DNA, a small but definite peak of 32p counts was observed around the region corresponding to the density of DNA. No such 32P-labelled peak was observed with heat-denatured E. coli DNA. The results indicate that the [32P]RNA preparation contains RNA which can form a specific hybrid with T4 DNA, but no detectable amount of RNA which can form a hybrid with E. coli DNA. The amount of
530
K.
OKA~IOTO,
Y . SlJG INO AXD l\L NOMURA
[S2P]RNA which formed a complex: with T4 DNA was very small, only about 1'5 % of the total [32P]RNA in this experiment. H owever, this is what would be expected, since t he [32P]RNA preparation used consists mostly of ribosomal and soluble RNA. Next, the experiments were performed at 25°0, to r educe the rate of synthesis of ribosomal and soluble RNA. OM was added 6 minutes before infection and 32p was added 4 minutes after infe ction. Assimilation of 32p was now st opped 40 seconds later . Control experiments without phage infection (E. coli messenger RNA) and witliout chloromycetin (phage messenger RNA) were also performed. As described in Table 1, pulse-labelled RNA, synthesized in OM-pretreated, T4-infected cells, showed the base composition typical of phage messenger RNA and different from that of host messenger RNA . A pulse-lab elled RNA preparation was also subjected to hybridization with heat-denatured T4 DNA and heat-denatured E . coli DNA, respectively . 1
0'2
(a)
\ I
\ ..- OoD. \
0'1
100
"... 10
20
30
• \s \ ~ 0·3
(b )
I
0
i§ 0·2 ci
C 0'1
200
~_on \,
31p _
'.
400 300
200 100
'0• •
~-
10
.S
~v
. ..~"'. 20
10 Fract ionnumber -
FIG. 1. Equilibrium density gradient centrifugation in CsCI. (a) A mixture of he at -denatured T4 D~A and S'p pulse -labe lled RXA. (b ) A mixture of heat -denatured E . coli DXA a nd u p pulselabe lled RNA. (c) A mixturo of heat -denatured E. coli DXA and [3'P]RKA from an E. coli "stepdown culture". ··P pulse-labelled RNA was prepared from CM-pretreated T4-infected cells wh ich assimilated "p for 2 min at 25°C from 4 to 6 min after infection. This RNA preparation (conta ining 170 p.g cold RNA) was u sed for the experiments (a ) and (b) . A [32P]RNA preparation (containing 180 p.g of cold RNA) prepared from a "stop-down culture" was used in experiment (c). Hybrid formation and the subsequent density gradient cen t rifugat .ion were performed as described in Materials and Methods.
About 25% of total [32P]RNA complexcd with heat-denatured T4 DNA (Fig . l(a» but none with heat-denatured E . coli DNA (Fig. l (b» . The same E. coli DNA preparation as used in these exp eriments was shown to form hybrid DNA quite efficiently with E. coli messenger RNA (Fig . l(c». It is concluded that messenger RNA synthesized in cells treated with OM before infection is mostly, if not all , phage messenger RNA. Since the amount of radioact ive
T4 MESSENGER RNA AFTER CHLOROMYCETIN TREATMENT
531
pulse RNA synthesized in CM-pretreated phage-infected cells is about one-third to one-half of that in CM-treated uninfected cells, and it is mostly phage messenger RNA, the synthesis of host messenger RNA appears to be inhibited almost completely. On the other hand, the synthesis of a considerable amount of host ribosomal and soluble RNA is demonstrated under the same conditions. (b) Turnover of phage messenger RNA in the presence of chloromycetin
Astrachan & Volkin (1959) studied the turnover of RNA synthesized in T2infected E. coli in the presence of CM. They concluded that, if the infected cells were inhibited with CM before or shortly after infection, newly synthesized RNA did not "turnover" and phage DNA was not synthesized. When CM was added 9 minutes
co
~ ~
:r: M
400
(bJ
300 200
200
20
40
Fraction number ___
FIG. 2. Conversion of pulse-labelled RNA (phage messenger RNA) to host ribosomal and soluble RNA. (a) Sedimentation of pulse-labelled [3H]RNA, synthesized in CM-pretreated T4-infected cells. (b) Sedimentation of [3H]RNA isolated after 20 min incubation with cold excess uracil. E. coli B6 was treated with CM 5 min before infection, and then infected with T4 at 37°C. 5 min after infection, cells were transferred to a 25°0 bath and incubated for an additional 5 min. [3H]uridine (300 mc/m-mole, final concentration 0·5 JLg/ml.) was supplied for 40 sec, and a portion was immediately chilled and RNA was prepared (samples for experiment (a)). To the remaining culture a 400·fold excess of both cold uridine and cytidine was added, and the culture was incubated for an additional 20 min at 37° C. RNA was then isolated (sample for experiment (b)). A [3H]RNA preparation was mixed with 3'P·labelled E. coli RNA containing both ribosomal RNA (16 s and 23 s) and soluble RNA (4 s), and analysed by the sucrose density gradient sedimentation technique. A 0·3 ml. sample was layered on top of a 4·4 ml. sucrose gradient (5 to 20%) containing 10-' M-tris (pH 7,2) and 0·05 M.KCl, but without MgH. Centrifugation was performed at 36,000 rev.rmin for 6 hr.
after infection, a condition that allows phage DNA synthesis, newly synthesized RNA did turn over. Thus, they correlated the RNA turnover with phage DNA synthesis. However, when CM was added before or shortly after infection, synthesis of host ribosomal and soluble RNA is dominant. Absence of turnover of phage messenger RNA under such conditions cannot be concluded from their experiments. As described in the previous section, when CM was added before infection, radioactive pulse RNA was phage messenger RNA, whereas long-time exposure of infected cells
532
K. OKAMOTO, Y. SUGINO AND M. NOMURA
to the radioactive precursor showed host ribosomal and soluble RNA as the main components of newly synthesized radioactive RNA. This fact itself suggests the turnover of phage messenger RNA under this condition. The following experiment supported this argument directly. A culture of E. coli B6 was treated with OMfor 5 minutes and then infected with T4 as described in Materials and Methods. [3H]uridine was supplied for 40 seconds at 25°0 and a portion was immediately chilled and analysed. To the remaining culture, a 400-fold excess of both cold uridine and cytidine was added and the culture was incubated for an additional 20 minutes at 37°0. The amount of total radioactive RNA after chasing was about the same or slightly higher than the amount of radioactive RNA before chasing. This result itself is the same as that reported by Astrachan & Volkin (1959); no breakdown of radioactive RNA into acid soluble material was observed. However, the sedimentation analysis of isolated RNA before and after chase showed the conversion of pulse-labelled messenger RNA to both ribosomal and soluble RNA (Fig. 2(a) and (b)). Parallel experiments showed that the pulselabelled radioactive RNA was mostly phage messenger ~NA, as described in the previous section. It is clear that some of the phage messenger RNA, synthesized in the presence of OM, breaks down and is re-utilized for the synthesis of ribosomal and soluble RNA in the absence of detectable protein synthesis.
4. Discussion The effect of OMon the inhibition of host RNA synthesis by phage T4 is summarized in Table 2. The effect on DNA synthesis in infected cells is also included (Tomizawa & Sunakawa, 1956; Nomura, Matsubara, Okamoto & Fujimura, 1962). When OM is added before phage infection, synthesis of host ribosomal and soluble RNA is observed. The initial rate is about one-half to one-third the rate of uninfected cells. The synthesis of host messenger RNA and DNA, however, is mostly inhibited. The synthesis of phage messenger RNA, but not phage DNA, takes place under such conditions. Turnover of phage messenger RNA is first shown by Astrachan & Volkin (1958). They showed that phage messenger RNA was converted to phage DNA. This conversion probably occurs through the degradation of phage messenger RNA to acid soluble ribonucleotides, subsequent reduction to deoxyribonucleotides and their polymerization into DNA (Cohen, Barner & Lichtenstein, 1961). Upon infection an increased activity of a deoxyribonucleotide synthesizing system was observed (Cohen et al., 1961) as well as other enzymes concerned with phage DNA synthesis (Flaks, Lichtenstein & Oohen, 1959; Kornberg, Zimmerman, Kornberg & Josse, 1959). Our conclusion that phage messenger RNA is synthesized in OM-pretreated infected cells and turns over in the presence of OM does not contradict earlier observations made by Astrachan & Volkin (1959). When OM was added 9 minutes after infection, they showed that the RNA synthesized was phage messenger RNA, and this RNA was converted to phage DNA. When OM was added before infection, they did not observe the change in the amount of total newly synthesized [32P]RNA after the chase· with excess [31P]phosphate. Our results show that, under the latter condition, phage messenger RNA does turn over and is converted to host ribosomal and soluble RNA. Synthesis of phage-induced enzymes concerned with DNA synthesis seems to be responsible for the alternative way of utilization of degradation products of phage messenger RNA, and turnover of phage messenger RNA itself takes place irrespective
1'4 :\rE8SENGER RXA AFTER CHLOROl\1YCETIN THEATMENT
533
of the time of addition of CM. Thus, protein synthesis does not seem to be obligatory for the breakdown of messenger RNA. However, our experiments say nothing about the precise rate of turnover of phage messenger RNA in the presence of CM. In fact, the stimulation of the accumulation of phage messenger RNA by CM was observed when eM was added after infection (Astrachan & Volkin, 1959; Nomura, Okamoto & Asano, 1962). It is possible that the rate of breakdown of phage messenger RNA is slower in the presence of CM than that in its absence. TABLE
2
Synthesis of various nucleic acids in E. coli B infected by phage T4 (or T2) and effects of chloromycetin (eM) Synthesis of
host DNA
host messenger RNA
ribosomal RNA
soluble RNA
+ +
+ +
+
+
+ +
+
+
phage DXA
phage messenger RNA
Uninfected E. coli NoCM CM 1'4 infected E. coli NoCM CM before infection C:\1after infection
+ +
+ + +
+ means that the amount synthesized is the order of magnitude comparable to that by control culture. - means nearly complete inhibition. The conclusions are based upon the data described in this and other papers (Nomura, Okamoto & Asano, 1962; Nomura, Matsubara, Okamoto & Fujimura, 1962; T'omizawa & Sunakawa, 1956.) Although it may be further supposed that some of the early phage-induced enzymes concerned with the conversion of phage messenger RNA to phage DNA themselves are the key proteins responsible for the inhibition of host ribosomal and soluble RNA synthesis after infection, and it is possible to explain the accumulated experimental observations by this assumption, a demonstration must await further experiments. Although Astrachan & Volkin (1959) concluded that some protein synthesis must precede or accompany formation of phage messenger RNA, it is now clear that phage messenger RNA can be synthesized without detectable prior protein synthesis. Their conclusion was based upon the base composition analysis of total [32P]RNA, synthesized in CM-pretreated infected cells. Our experimental results show that such [32P]RNA preparation may have consisted mainly of newly synthesized host ribosomal and soluble RNA in addition to a small amount of phage messenger RNA, and the presence of phage messenger RNA could not be detected by the base composition analysis of total newly synthesized [32P]RNA under their experimental conditions. The induced synthesis of phage messenger RNA and the simultaneous suppression of host messenger RNA synthesis by phage without prior protein synthesis has also been shown in the experiments of Volkin (1960). "Using an amino acid-requiring mutant of E. coli, he demonstrated the synthesis of phage messenger RNA, under conditions where protein synthesis is suppressed by deprivation of a required amino acid.
534
K. OKA:\IOTO, Y. SUGIXO AND M. NO;\[URA
Amino acid deprivation in an amino acid-requiring mutant suppresses the synthesis of both ribosomal and soluble RNA, but not the synthesis of messenger RNA (Okamoto & Nomura, unpublished experiments). This is probably the reason why the synthesis of phage messenger RNA (detected by base composition analysis of L32P ]R NA obtained) could be seen after a fairly long exposure to 32 P, and was not masked by the synthesis of ribosomal and soluble RNA. This investigation was aided in part by research grant E-3809 from the National Institutes of Health, United States Public Health Service. REFERENCES Astrachan, L. & Volkin, E. (1958). Biochim. biophys. Acta, 29, 536. Astrachan, L. & Volkin, E. (1959). Biochim. biophys. Acta, 32, 449. Brenner, S., Jacob, F. & Meselson, M. (1961). Nature, 190, 576. Cohen, S. S., Barner, H. D. & Lichtenstein, J. (1961). J. BioI. Chem, 236, 1448. Dunn, D. B. & Smith, J". D. (1958). Biochem. J. 68, 627. Flaks, J. G., Lichtenstein, J. & Cohen, S. S. (1959). J. BioI. Chem. 234, 1507. Gros, F., Hiatt, H., Gilbert, W., Kurland, R. W., Risebrough, R. W. & Watson, J. D. (1961). Nature, 190, 581. Hall, B. D. & Spiegelman, S. (1961). Proc, Nat. Acad. Sci., Wash. 47, 137. Hayashi, M. & Spiegelman, S. (1961). Proc, Nat. Acad. Sci., Wash. 47, 1564. Kornberg, A., Zimmerman, S. B., Kornberg, S. R. & Josse, J. (1959). Proc. Nat. Acad. Sci., Wash. 45, 772. Mandell, J. D. & Hershey, A. D. (1960). Anal. Biochem. 1, 66. Marmur, J. (1961). J. Mol. ei«. 3, 208. Monier, R., Naono, S., Hayes, D., Hayes, F. & Gros, F. (1962). J. Mol. BioI. 5, 311. Nomura, M., Hall, B. D. & Spiegelman, S. (1960). J. Mol. BioI. 2, 306. Nomura, M., Matsubara, K., Okamoto, K. & Fujimura, R. (1962). J. Mol. Biol. 5, 535. Nomura, M., Okamoto, K. & Asano, K. (1962). J. Mol. Biol. 4, 376. Otaka, E., Mitsui, H. & Osawa, S. (1962). Proc. Nat. Acad. Sci., Wash. 48, 425. Sagik, B. P., Green, M. H., Hayashi, M. & Spiegelman, S. (1962). Biophys. J. in the press. Spahr, P. F. & Tissieres, A. (1959). J. Mol. Biol. 1, 237. Tomizawa, J. & Sunakawa, S. (1956). J. Gen. Physiol. 39, 553. Volkin, E. (1960). Proc. Nat. Acad. Sci., Wash. 46, 1336. Volkin, E. & Astrachan, L. (1956). Virology, 2, 149. Wyatt, G. (1953). Cold Spr, Harb. Symp. Quant. Bioi. 18, 133.
Synthesis and Turnover of Phage Messenger RNA in E. Coli Infected with Bacteriophage T4 in the Presence of Chloromycetin K OJI OKAMOTO ,
Y.
S u aINO AND M ASAYA SU NO:MURA
Lnstituie fo r P rotein R esearch, Osaka University, Osaka, Japan (R eceived 13 August 1962) Treatment of E. coli with ch loro m ycet in prior to infection with T4 allows both ribosomal RNA and soluble RNA to be synthesized after infection. Both base composition analysis and hybridization experiments with DNA showed that the majority, if not all, of the pulse-labelled RNA synthesized in CM-pretreated, T4-infected cells was phage messenger RNA. The synthesis of host messenger RNA is inhibited by phage under suc h conditions, as is the synt h esis of host DNA. Phage messen ger RNA synthes ized in CM-pretreated , T4.infeeted cells was shown to be d egraded and repolymerized into ribosomal RNA and soluble RNA in the presence of CM.
1. Introduction Although after T-even phage infection the synthesis of typical ribosomal and soluble RNA st ops , a small am ount of phage-specific RNA is synthesized (Volkin & Astrachan, 1956 ; Nomura, H all & Spiegelman, 1960; Brenner, J acob & Meselson, 1961). It is suggested that this "messenger" RNA carries the information for the proteins synthesized after ph age infection. A similar kind of RNA is also found in uninfected cells (Gros ~ al., 1961 ; Hayashi &Spiegelman, 1961). Mter phage infection very little host messenger RNA is synthesized and the inhibition of host protein synthesis by ph age seems t o be du e to the inhibition of host messenger RNA synthesis. In a previous paper (Nomura, Okamoto & Asan o, 1962) we have shown that treatment of E. coli with ohloromycetirr] prior to infection with T4 allows both ribosomal RNA and soluble RNA to be synthesized after infection. It was suggested that, upon infection, the phage DNA induces the synthesis of some specific protein which causes the inhibition of the synthesis of host ribosomal and soluble RNA. lithe inhibition of the synthesis of host messenger RNA by phage is due to the same mechanism as that of ribosomal and soluble RNA, it would be expected that the addition of CM before infe ction would also prevent the inhibition of host messenger RNA synt hesis. The experimental results pre sented in this paper show that, on the contra ry, this inhibition still occurs in the presence of CM. The majority, if not all , of the messenger RNA synthesized in CM-pretreated, T4-infected cells is phage messenger RNA. It thus appears that phage infection can cause the inhibition of host messenger RNA syn t hesis without first producing a protein. As a consequ ence of this study, turnover of phage messenger RNA in infe cted cells in the presence of CM was presumed and , in fact, demonstrated.
t Abbreviation used:
chloromycetin =
eM. 527
.'i28
K. OKAMOTO, Y. Sl:GIKO AXD
~1.
XO~lURA
2. Materials and Methods E. coli strain B growing exponentially in tris-glueose-Casamino acids medium was infected with bacteriophage T4r+ in the presence of tryptophan (50fkg/ml.) at a multiplicity between 10 and 20, as described in a previous paper (Nomura, Okamoto & Asano, 1962). E. coli strain B6 (an auxotrophic mutant requiring both arginine and uracil) was used instead ofB when [3H]nridine was used to label RNA. Excess uracil (30fkg/ml.) was added to tris-glueose-Casamino acids medium to grow cells. Exponentially growing cells were collected by centrifugation at room temperature, washed with tris-glucose-Casamino acids medium (without uracil), and resuspended in the same medium (without uracil). Cells were infected with T4r+ as described above. R~A was isolated directly from cells without a prior grinding with alumina. The experiment was terminated by the addition of azide (10- 2 :'<1) and the culture was immediately poured onto crushed ice. Usually an appropriate amount of carrier uninfeeted cells (about 1011 cells) was added. The cells were then centrifuged and the pellets, without washing, were suspended in the cold in 3 m!. of 10- 2 ::II:-tris, [) x 10- 3 ::II:-Mgh, containing 10- 2 M-azide. Both D'Nase (20 fkg/ml.) and lysozyme (200 fkg/ml.) were added and the solution frozen in a dry-ice bath. After 3 freezings and thawings, the pH of the solution was brought to 5·2 by the addition of 5 drops of 0·02 M-CH 3COOH. The solution was then treated with sodium dodecylsulphate (recrystallized material, final concentration 0·3%) at 37°C for 3 min, and then shaken at 37°C for 5 min with an equal volume of phenol saturated with 0·05 ::II:-aeetate (pH 5,2)-0,01 M-Mg2+ solution. After centrifugation, RNA was precipitated with 2 vo!. alcohol, dissolved in 0·02 M-aeetate (pH 5,2) containing 0·02 M-KCI and 0·01 M-:\fg2+, and repreeipitated with alcohol. The final precipitate was dissolved in the same aeetate-KCI-MgCI 2 solution and dialysed overnight against 200 vol. of the same solution in the cold, with several changes of outer solution. With the present technique we consistently obtained phage messenger RNA with a sedimentation coefficient considerably higher than the value of 8 s reported in earlier papers (~omura et al., 1960; Gros et al., 1961). An example of a sedimentation pattern of phage messenger RNA obtained by this technique is shown in Fig. 2(a). This result is in accord with the recent results obtained in several other laboratories (Otaka, Mitsui & Osawa, 1962; Monier, Naono, Hayes, Hayes & Gros, 1962; Sagik, Green, Hayashi & Spiegelman, 1962). Hybrid formation of messenger RXA with heat-denatured DNA and the equilibrium density gradient centrifugation in CsCI were performed in a manner similar to that described by Hall & Spiegelman (1961). Heat-denatured D~A was prepared by heating a solution of DNA at 100°C for 15 min and then cooling rapidly in an icc bath. A UP-labelled RKA preparation was mixed with 10 fkg of heat-denatured DKA in 0·5 ml. of 0·3 M-~aCI 0·03 M-sodium citrate solution and incubated at 40°C for 36 hr. Ten fkg of heat-denatured DNA was then added and the mixture made up to 4 ml. with CsCI solution, such that the final concentration of CsCI was 57% (w/w). The solutions were centrifuged at 33,000 rev./min near O°C for 60 hr in a swinging bucket rotor, SW39, in a Spinco model L ultracentrifuge. At the end of the run, the tube was pierced with a needle and fractions of 10 drops each were collected. After suitable dilution of the fractions, ultraviolet absorption at 260 mfk and acid precipitable S2p of each fraction were determined. Phage 1'4 DNA was prepared according to Mandell & Hershey (1960). E. coli DNA was prepared by the method of Marmur (1961). E. coli "messenger RNA" used as a reference standard in the experiment of Fig. 1 was prepared from a "step-down culture" (Hayashi & Spiegelman, 1961). Cells were grown in modified Pcnassey-broth (Nomura et al., 1960) to a titre of 3 x 108/ml., harvested, washed and suspended in the tris-glueose synthetic medium (without Casamino acids). [32P]orthophosphate was then added for 5 min at 37°C. Cells were quickly chilled and RNA was isolated as described above. Sedimentation analyses of 3H-labelled RNA in sucrose density gradient with 32P-labelled RKA as sedimentation markers are performed as described in the following paper (Nomura, Matsubara, Okamoto & Fujimura, 1962). The analysis of RNA base composition was described in previous papers (Nomura et al., 1960; Xomura, Okamoto & Asano, 1962).
T4 MESSENGER RNA AFTER CHLOROMYCETIN TREATM:ENT
529
3. Results (a) Nature of messenger RNA synthesized in cells treated with OM before infection
In our previous paper, characterization of ribosomal and soluble RNA synthesized in eM-pretreated infected cells was described (Nomura, Okamoto & Asano, 1962). In order to characterize the messenger RNA, two kinds of techniques were used. One involves the formation of specific DNA-RNA hybrids. The other is the determination of the base composition of the [32P]RNA synthesized after a short exposure to [32P]orthophosphate at low temperature. Under such conditions, the newly synthesized [32P]RNA is mostly messenger RNA (Gros et al., 1961). TABLE
1
Base composition of 32 P pulse-labelled RNA synthesized in T4-infected cells treated with chloromycetin (OM) before infection s2P-pulse
o sec
CM: (before infection)
at 25°C
10 min at 37°Ct phage DNAt E. coli DNA§ E. coli ribosomal RNA'\[
+ + +
Base composition (A = 1,00)
Phage C
A
G
U
+ +
0·60 0·59 0·96
1·00 1·00 1·00
0·81 0·82 1·25
1·10 H2 1·15
+
0·85
1·00
1·27
0·83
0·53 1·09 0·88
1·00 1·00 1·00
0·56 1·14 1·26
1·0011 1·0811 0·86
Culture of E. coli B exponentially growing in tris-glucose-Casamino acids medium at 37°C (2 x lOs/mI.) was transferred to a 25°C bath and incubated for 10 min. The culture was then treated with CM: (100 f.Lg/mI.) and, 6 min later, infected with T4 at a multiplicity of 10 in the presence of tryptophan (50 f.Lg/mI.). [32P]orthophosphate was added 5 min after infection. Assimilation of 32p was stopped 40 sec later. Control cultures without either CM: or phage infection were also analysed.
t Data published in a previous paper (Nomura, Okamoto & Asano, 1962). t Wyatt (1953). § Dunn & Smith (1958). 'II Spahr & Tissieres (1959). II Value for thymidylic acid instead of uridylic acid. First, experiments were performed at 37°C as previously described (Nomura, Okamoto & Asano, 1962). CM (80 JLg/ml.) was added 6 minutes before infection and [32P]orthophosphate was supplied between 4 and 14 minutes after infection. The entire culture was then chilled and RNA was prepared. This preparation was shown to contain both ribosomal RNA (16 sand 23 s) and soluble RNA (4 s) as the major 32P-Iabelled components. This preparation was subjected to the hybridization process with heat-denatured T4 DNA and with heat-denatured E. coli DNA. With heatdenatured phage DNA, a small but definite peak of 32p counts was observed around the region corresponding to the density of DNA. No such 32P-labelled peak was observed with heat-denatured E. coli DNA. The results indicate that the [32P]RNA preparation contains RNA which can form a specific hybrid with T4 DNA, but no detectable amount of RNA which can form a hybrid with E. coli DNA. The amount of
530
K.
OKA~IOTO,
Y . SlJG INO AXD l\L NOMURA
[S2P]RNA which formed a complex: with T4 DNA was very small, only about 1'5 % of the total [32P]RNA in this experiment. H owever, this is what would be expected, since t he [32P]RNA preparation used consists mostly of ribosomal and soluble RNA. Next, the experiments were performed at 25°0, to r educe the rate of synthesis of ribosomal and soluble RNA. OM was added 6 minutes before infection and 32p was added 4 minutes after infe ction. Assimilation of 32p was now st opped 40 seconds later . Control experiments without phage infection (E. coli messenger RNA) and witliout chloromycetin (phage messenger RNA) were also performed. As described in Table 1, pulse-labelled RNA, synthesized in OM-pretreated, T4-infected cells, showed the base composition typical of phage messenger RNA and different from that of host messenger RNA . A pulse-lab elled RNA preparation was also subjected to hybridization with heat-denatured T4 DNA and heat-denatured E . coli DNA, respectively . 1
0'2
(a)
\ I
\ ..- OoD. \
0'1
100
"... 10
20
30
• \s \ ~ 0·3
(b )
I
0
i§ 0·2 ci
C 0'1
200
~_on \,
31p _
'.
400 300
200 100
'0• •
~-
10
.S
~v
. ..~"'. 20
10 Fract ionnumber -
FIG. 1. Equilibrium density gradient centrifugation in CsCI. (a) A mixture of he at -denatured T4 D~A and S'p pulse -labe lled RXA. (b ) A mixture of heat -denatured E . coli DXA a nd u p pulselabe lled RNA. (c) A mixturo of heat -denatured E. coli DXA and [3'P]RKA from an E. coli "stepdown culture". ··P pulse-labelled RNA was prepared from CM-pretreated T4-infected cells wh ich assimilated "p for 2 min at 25°C from 4 to 6 min after infection. This RNA preparation (conta ining 170 p.g cold RNA) was u sed for the experiments (a ) and (b) . A [32P]RNA preparation (containing 180 p.g of cold RNA) prepared from a "stop-down culture" was used in experiment (c). Hybrid formation and the subsequent density gradient cen t rifugat .ion were performed as described in Materials and Methods.
About 25% of total [32P]RNA complexcd with heat-denatured T4 DNA (Fig . l(a» but none with heat-denatured E . coli DNA (Fig. l (b» . The same E. coli DNA preparation as used in these exp eriments was shown to form hybrid DNA quite efficiently with E. coli messenger RNA (Fig . l(c». It is concluded that messenger RNA synthesized in cells treated with OM before infection is mostly, if not all , phage messenger RNA. Since the amount of radioact ive
T4 MESSENGER RNA AFTER CHLOROMYCETIN TREATMENT
531
pulse RNA synthesized in CM-pretreated phage-infected cells is about one-third to one-half of that in CM-treated uninfected cells, and it is mostly phage messenger RNA, the synthesis of host messenger RNA appears to be inhibited almost completely. On the other hand, the synthesis of a considerable amount of host ribosomal and soluble RNA is demonstrated under the same conditions. (b) Turnover of phage messenger RNA in the presence of chloromycetin
Astrachan & Volkin (1959) studied the turnover of RNA synthesized in T2infected E. coli in the presence of CM. They concluded that, if the infected cells were inhibited with CM before or shortly after infection, newly synthesized RNA did not "turnover" and phage DNA was not synthesized. When CM was added 9 minutes
co
~ ~
:r: M
400
(bJ
300 200
200
20
40
Fraction number ___
FIG. 2. Conversion of pulse-labelled RNA (phage messenger RNA) to host ribosomal and soluble RNA. (a) Sedimentation of pulse-labelled [3H]RNA, synthesized in CM-pretreated T4-infected cells. (b) Sedimentation of [3H]RNA isolated after 20 min incubation with cold excess uracil. E. coli B6 was treated with CM 5 min before infection, and then infected with T4 at 37°C. 5 min after infection, cells were transferred to a 25°0 bath and incubated for an additional 5 min. [3H]uridine (300 mc/m-mole, final concentration 0·5 JLg/ml.) was supplied for 40 sec, and a portion was immediately chilled and RNA was prepared (samples for experiment (a)). To the remaining culture a 400·fold excess of both cold uridine and cytidine was added, and the culture was incubated for an additional 20 min at 37° C. RNA was then isolated (sample for experiment (b)). A [3H]RNA preparation was mixed with 3'P·labelled E. coli RNA containing both ribosomal RNA (16 s and 23 s) and soluble RNA (4 s), and analysed by the sucrose density gradient sedimentation technique. A 0·3 ml. sample was layered on top of a 4·4 ml. sucrose gradient (5 to 20%) containing 10-' M-tris (pH 7,2) and 0·05 M.KCl, but without MgH. Centrifugation was performed at 36,000 rev.rmin for 6 hr.
after infection, a condition that allows phage DNA synthesis, newly synthesized RNA did turn over. Thus, they correlated the RNA turnover with phage DNA synthesis. However, when CM was added before or shortly after infection, synthesis of host ribosomal and soluble RNA is dominant. Absence of turnover of phage messenger RNA under such conditions cannot be concluded from their experiments. As described in the previous section, when CM was added before infection, radioactive pulse RNA was phage messenger RNA, whereas long-time exposure of infected cells
532
K. OKAMOTO, Y. SUGINO AND M. NOMURA
to the radioactive precursor showed host ribosomal and soluble RNA as the main components of newly synthesized radioactive RNA. This fact itself suggests the turnover of phage messenger RNA under this condition. The following experiment supported this argument directly. A culture of E. coli B6 was treated with OMfor 5 minutes and then infected with T4 as described in Materials and Methods. [3H]uridine was supplied for 40 seconds at 25°0 and a portion was immediately chilled and analysed. To the remaining culture, a 400-fold excess of both cold uridine and cytidine was added and the culture was incubated for an additional 20 minutes at 37°0. The amount of total radioactive RNA after chasing was about the same or slightly higher than the amount of radioactive RNA before chasing. This result itself is the same as that reported by Astrachan & Volkin (1959); no breakdown of radioactive RNA into acid soluble material was observed. However, the sedimentation analysis of isolated RNA before and after chase showed the conversion of pulse-labelled messenger RNA to both ribosomal and soluble RNA (Fig. 2(a) and (b)). Parallel experiments showed that the pulselabelled radioactive RNA was mostly phage messenger ~NA, as described in the previous section. It is clear that some of the phage messenger RNA, synthesized in the presence of OM, breaks down and is re-utilized for the synthesis of ribosomal and soluble RNA in the absence of detectable protein synthesis.
4. Discussion The effect of OMon the inhibition of host RNA synthesis by phage T4 is summarized in Table 2. The effect on DNA synthesis in infected cells is also included (Tomizawa & Sunakawa, 1956; Nomura, Matsubara, Okamoto & Fujimura, 1962). When OM is added before phage infection, synthesis of host ribosomal and soluble RNA is observed. The initial rate is about one-half to one-third the rate of uninfected cells. The synthesis of host messenger RNA and DNA, however, is mostly inhibited. The synthesis of phage messenger RNA, but not phage DNA, takes place under such conditions. Turnover of phage messenger RNA is first shown by Astrachan & Volkin (1958). They showed that phage messenger RNA was converted to phage DNA. This conversion probably occurs through the degradation of phage messenger RNA to acid soluble ribonucleotides, subsequent reduction to deoxyribonucleotides and their polymerization into DNA (Cohen, Barner & Lichtenstein, 1961). Upon infection an increased activity of a deoxyribonucleotide synthesizing system was observed (Cohen et al., 1961) as well as other enzymes concerned with phage DNA synthesis (Flaks, Lichtenstein & Oohen, 1959; Kornberg, Zimmerman, Kornberg & Josse, 1959). Our conclusion that phage messenger RNA is synthesized in OM-pretreated infected cells and turns over in the presence of OM does not contradict earlier observations made by Astrachan & Volkin (1959). When OM was added 9 minutes after infection, they showed that the RNA synthesized was phage messenger RNA, and this RNA was converted to phage DNA. When OM was added before infection, they did not observe the change in the amount of total newly synthesized [32P]RNA after the chase· with excess [31P]phosphate. Our results show that, under the latter condition, phage messenger RNA does turn over and is converted to host ribosomal and soluble RNA. Synthesis of phage-induced enzymes concerned with DNA synthesis seems to be responsible for the alternative way of utilization of degradation products of phage messenger RNA, and turnover of phage messenger RNA itself takes place irrespective
1'4 :\rE8SENGER RXA AFTER CHLOROl\1YCETIN THEATMENT
533
of the time of addition of CM. Thus, protein synthesis does not seem to be obligatory for the breakdown of messenger RNA. However, our experiments say nothing about the precise rate of turnover of phage messenger RNA in the presence of CM. In fact, the stimulation of the accumulation of phage messenger RNA by CM was observed when eM was added after infection (Astrachan & Volkin, 1959; Nomura, Okamoto & Asano, 1962). It is possible that the rate of breakdown of phage messenger RNA is slower in the presence of CM than that in its absence. TABLE
2
Synthesis of various nucleic acids in E. coli B infected by phage T4 (or T2) and effects of chloromycetin (eM) Synthesis of
host DNA
host messenger RNA
ribosomal RNA
soluble RNA
+ +
+ +
+
+
+ +
+
+
phage DXA
phage messenger RNA
Uninfected E. coli NoCM CM 1'4 infected E. coli NoCM CM before infection C:\1after infection
+ +
+ + +
+ means that the amount synthesized is the order of magnitude comparable to that by control culture. - means nearly complete inhibition. The conclusions are based upon the data described in this and other papers (Nomura, Okamoto & Asano, 1962; Nomura, Matsubara, Okamoto & Fujimura, 1962; T'omizawa & Sunakawa, 1956.) Although it may be further supposed that some of the early phage-induced enzymes concerned with the conversion of phage messenger RNA to phage DNA themselves are the key proteins responsible for the inhibition of host ribosomal and soluble RNA synthesis after infection, and it is possible to explain the accumulated experimental observations by this assumption, a demonstration must await further experiments. Although Astrachan & Volkin (1959) concluded that some protein synthesis must precede or accompany formation of phage messenger RNA, it is now clear that phage messenger RNA can be synthesized without detectable prior protein synthesis. Their conclusion was based upon the base composition analysis of total [32P]RNA, synthesized in CM-pretreated infected cells. Our experimental results show that such [32P]RNA preparation may have consisted mainly of newly synthesized host ribosomal and soluble RNA in addition to a small amount of phage messenger RNA, and the presence of phage messenger RNA could not be detected by the base composition analysis of total newly synthesized [32P]RNA under their experimental conditions. The induced synthesis of phage messenger RNA and the simultaneous suppression of host messenger RNA synthesis by phage without prior protein synthesis has also been shown in the experiments of Volkin (1960). "Using an amino acid-requiring mutant of E. coli, he demonstrated the synthesis of phage messenger RNA, under conditions where protein synthesis is suppressed by deprivation of a required amino acid.
534
K. OKA:\IOTO, Y. SUGIXO AND M. NO;\[URA
Amino acid deprivation in an amino acid-requiring mutant suppresses the synthesis of both ribosomal and soluble RNA, but not the synthesis of messenger RNA (Okamoto & Nomura, unpublished experiments). This is probably the reason why the synthesis of phage messenger RNA (detected by base composition analysis of L32P ]R NA obtained) could be seen after a fairly long exposure to 32 P, and was not masked by the synthesis of ribosomal and soluble RNA. This investigation was aided in part by research grant E-3809 from the National Institutes of Health, United States Public Health Service. REFERENCES Astrachan, L. & Volkin, E. (1958). Biochim. biophys. Acta, 29, 536. Astrachan, L. & Volkin, E. (1959). Biochim. biophys. Acta, 32, 449. Brenner, S., Jacob, F. & Meselson, M. (1961). Nature, 190, 576. Cohen, S. S., Barner, H. D. & Lichtenstein, J. (1961). J. BioI. Chem, 236, 1448. Dunn, D. B. & Smith, J". D. (1958). Biochem. J. 68, 627. Flaks, J. G., Lichtenstein, J. & Cohen, S. S. (1959). J. BioI. Chem. 234, 1507. Gros, F., Hiatt, H., Gilbert, W., Kurland, R. W., Risebrough, R. W. & Watson, J. D. (1961). Nature, 190, 581. Hall, B. D. & Spiegelman, S. (1961). Proc, Nat. Acad. Sci., Wash. 47, 137. Hayashi, M. & Spiegelman, S. (1961). Proc, Nat. Acad. Sci., Wash. 47, 1564. Kornberg, A., Zimmerman, S. B., Kornberg, S. R. & Josse, J. (1959). Proc. Nat. Acad. Sci., Wash. 45, 772. Mandell, J. D. & Hershey, A. D. (1960). Anal. Biochem. 1, 66. Marmur, J. (1961). J. Mol. ei«. 3, 208. Monier, R., Naono, S., Hayes, D., Hayes, F. & Gros, F. (1962). J. Mol. BioI. 5, 311. Nomura, M., Hall, B. D. & Spiegelman, S. (1960). J. Mol. BioI. 2, 306. Nomura, M., Matsubara, K., Okamoto, K. & Fujimura, R. (1962). J. Mol. Biol. 5, 535. Nomura, M., Okamoto, K. & Asano, K. (1962). J. Mol. Biol. 4, 376. Otaka, E., Mitsui, H. & Osawa, S. (1962). Proc. Nat. Acad. Sci., Wash. 48, 425. Sagik, B. P., Green, M. H., Hayashi, M. & Spiegelman, S. (1962). Biophys. J. in the press. Spahr, P. F. & Tissieres, A. (1959). J. Mol. Biol. 1, 237. Tomizawa, J. & Sunakawa, S. (1956). J. Gen. Physiol. 39, 553. Volkin, E. (1960). Proc. Nat. Acad. Sci., Wash. 46, 1336. Volkin, E. & Astrachan, L. (1956). Virology, 2, 149. Wyatt, G. (1953). Cold Spr, Harb. Symp. Quant. Bioi. 18, 133.