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Regulation of early RNA synthesis in bacteriophage T4-infected Escherichia coli cells
J. Mol. Biol. (1970) 53, 339-356
Regulation of Early RNA Synthesis in Bacteriophage T&infected Escherichia coli Cells OLA SK~LD Department
of Microbiology,
Faculty
of Pharmacy
University of Uppsala, Xweden (Received 22 April
1970, and in revised form 9 July 1970)
The regulation of synthesis was examined for those early mRNA species in bacteriophage T4 infection which are responsible for the formation of early enzymes involved in DNA synthesis. Evidence was found for a transcriptional regulation mechanism, connected to the function of phage genes 33 and 55. Early mRNA formation was observed to be sharply turned off at about the time of normal initiation of DNA synthesis. This distinct shut-off was abolished if the infecting phage contained a lesion in gene 33 or 55. The determination of synthesis of those early mRNA species responsible for phage DNA formation was based on the observation that the presence of 5-fluorouraoil made the formation of phage DNA heat-sensitive. This was interpreted as a consequence of fluorouracil incorporation into early mRNA, where it introduces coding errors, which in turn cause heat sensitivity of the phage-specific, DNAforming enzymes. The fluorouracil inhibition of phage DNA synthesis could be reversed either by a shift-down in temperature or by the addition of a high concentration of uridine. In the latter case the efficiency of rescue decreased rapidly with time of uridine addition after infection. This is thought to reflect the formation of those mRNA species which are responsible for the synthesis of DNAforming enzymes. Uridine could chase out fluorouracil from early mRNA and allow synthesis of error-free messenger and subsequently of heat-stable early enzymes, but only as long as the synthesis of these particular mRNA species proceeded. The turn-off of early mRNA synthesis and its dependence on an undisturbed gene 55 function could also be demonstrated by short-time labelling of total RNA, which formed after infection with early mutants producing only early mRNA. In this case the uptake of labelled precursor into nucleotide pools at different times after infection was checked by electrophoretic analysis.
1. Iutroduction For many years it has been well established, that when a T-even phage, such as T4, infects a sensitive Escherichia coli cell, host macromolecular synthesis stops immediately and RNA, DNA and proteins controlled by the viral genome start to be synthesized (Cohen, 1948; Nomura, Okamoto & Asano, 1962; Nomura, Witten, Mantei & Echols, 1966). In the early period after infection many phage-specific proteins are synthesized which will not be incorporated in the mature phage. Many of these early proteins have been demonstrated to be enzymes involved in the biosynthesis of phage DNA (Cohen, 1963, 1968). Most of the proteins formed during the later period after infection become 339
340
0.
SKOLI)
part of the mature phage as structural proteins. The late pathlrays of assembly ar(s under investigation (Edgar & Wood, 1966 ; King & Wood, 1969). Results of Hosoda & Levinthal (1968) show that this simple classification of phage proteins into early and late is not adequate. By protein labelling, disc elcctrophoresis and autoradiography they have distinguished a group of phage proteins, the synthesis of which begins much more rapidly after infection t’han does synthesis of most of the early proteins. The class of early phage proteins is thus not controlled as a single homogeneous group, but synthesis of its members is both initiated and shut ofI’ at various times during the early part of the infect,ion process. The best-characterized of the early proteins arc those involved in phage DNA synthesis (Cohen, 1968) and for this group of proteins, there seems to exist a co-ordinate regulation mechanism, which shuts off net synthesis of enzymes at a definite time after infection (Kornberg, Zimmerman, Kornberg & Jossc, 1959; Wiberg, Dirksen, Epstein, Luria & Buchanan, 1962). The general features of the temporal sequence of phage protein synthesis are reflected in the changing pattern of hybridizable RNA, pulse-labelling in phage-infected cells (Khesin, Gorlenko, Shemyakin, Bass & Prozorov, 1963; Hall, Nygaard & Green, 1964). Furthermore Belle, Epstein, Salser & Geiduschek (1968a) could show by the refined technique of RNA-DNA hybridization-competition, that at 20 minutes after infection, at 30°C “late” RNA species were several hundredfold more abundant than at five minutes after infection, and in a later work (Salser, Bolle & Epstein, 1970) they showed that “true-early” RNA decreased severalfold in level between 5 and 20 minutes after infection. They concluded that these observations reflected changing rates of transcription. The experiments described in this paper aim at studying the regulation of synthesis, specifically of those mRNA species responsible for the formation of early enzymes involved in phage-specific DNA synthesis. The experimental approach was based on the observation, that the presence of 5-fluorouracil in the medium made phage production, and more specifically phage DNA synthesis heat-sensitive. Both DNA synthesis and phagc production could, however, be rescued at the high temperature by the addition of uridine. The efficiency of this uridine rescue rapidly diminished wit,h time after infection. The inhibition of phagc DNA synthesis by FUt (in the presence of thymidine) was explained by its incorporation into early mRNA, thus inducing coding errors and making phage enzymes involved in DNA synthesis heat-sensitive. The rescue from the FU inhibition by uridine would then take place by the chasing out of FU, but only as long as active synthesis of early mRNA proceeded. The observed decrease in rescue efficiency with time after infection is interpreted to reflect a normal regulation of early RNA synthesis. Mut,ations in genes 33 and 55 were found to interfere with this regulation.
2. Materials and Methods (a) Bacteria
and Bacteriophage
E. coli B and a uraoil-requiring mutant of it (OK 302, Karlstrom, 1968) was used. Wild type bacteriophage T4D and mutant rcmC42 (gene 1, hydroxymethyl deoxycytidylate kinase) was obtained from Dr J. S. Wiberg. The T4 mutants amN134 (gene 33) and amBL292 (gene 55), were provided by Dr A. Belle. The double mutant amC42 x amBL292 was constructed and identified by the complement&ion spot test technique t Abbreviations
and triphosphate,
used:
FU,
respectively.
5-fluoroaracil;
FUMP,
FUDP,
FUTP,
5.fluorouridine
mono-,
di-
REGULATION described by Wiberg dz Buchanan, 1964).
(1966). Phages
OF T4 RNA were grown
341
SYNTHESIS
and harvested
as described
earlier
(Skolci
(b) ChernicaLs [MethyZ-3H] thymidine (2.0 to 5.0 c/m-mole) and [6-3H]5-fluorouracil ~870 me/m-mole) was obtained from The Radiochemical Centre, Amersham, England. ~-[~~C]Leucine (uniformly labelled, 250 me/m-mole) was purchased from New England Nuclear Chemicals, Dreieichenhain, Germany and 2-14C-labelled 5-fluorouracil (22 me/m-mole) from Calbiothem, Luzern, Switzerland. Unlabelled 5fluourouracil was a gift from F. Hoffman-La Roche & Co., Basle, Switzerland. (c) Media All experiments were carried out in a low phosphate mineral salts medium of the following composition/l.: 0.5 g NH,Cl, 0.5 g (NH4)zS04. 5.0 g NaCl, 0.5 g KCl, 0.203 g MgCl,.6H,O, 14.7 mg CaCl,.2H,O, 0.178 g Na2HP04.2H,0 and 6.25 g Tris, pH was adjusted to 8.0 with M-HCl and after amoclaving the medium was supplemented with glucose 5 g/l. and FeCI, to 10m5 r+r, Bottom agar for phage plating, contained/l.: 10 g Agar (Bacto), 13 g Bactotryptone, 8 g NaCl, 2.3 g sodium citrate.2H,O, 1.3 g glucose. Top agar for phage plating contained/l.: 6 g Agar (Bacto), 10 g Bactotryptone, 8 g NaCl, 2.3 g sodium citrate.2Ha0, 3 g glucose, 20 mg L-tryptophan. Dilution fluid for phage contained/l. : 10 g Bactotryptone, 5 g Nacl, 20 mg L-tryptophan. (d) Infected
cells
Bacteria were grown to 5 x lO*/ml. at 37°C in the mineral salts medium, (supplemented with uridine 10 pg/ml. for OK302, generation time about 53 mm). The harvested cells were washed once and resuspended to about 1.5 x 1 Og/ml. in the same medium supplement& with L-tryptophan 10 pg/ml., thymidine 20 pg/ml. and FU 10 pg/ml. or uridine 10 pg/ml. Infection was performed by the addition of 7 to 9 phages per bacterium. After 3 min of adsorption the infected cells were diluted (usually lo-fold) into warmed, aerated mineral salts medium containing radioisotope and FU or uridine. All experiments with FU were performed in the presence of thymidine to neutralize the very strong inhibitory effect on thymidylate synthetase by 5-fluorodeoxyuridine monophosphate, which could form intracellularly (Cohen, Flaks, Barrier, Loeb & Lichtenstein, 1958). Phage adsorption, bacterial survival, number of infective centres and burst size were determined by standard techniques. Adsorption was usually better than 99% at the end of the 3-min adsorption period and bacterial survival less than 1 ?A. In all experiments phage infection is coumed as time zero. (e) Measurement
of RNA,
DLNA and protein
synthesis
The synthesis of DNA and protein was determined by the cumulative incorporation of [3H:]thymidine and L-[%]leucine, respectively, into acid-insoluble product. Similarly the incorporation of FU into RNA was studied by the cumulative incorporation of 14C- or 3H-labeled 5-fluorouracil into acid-insoluble product. In the latter case there would presumably be no interference from FU being incorporated into phage DNA, since that would require the removal of the &jZuorine, and 5-fluorouracil is known to be quite stable intracellularly (Horowitz & Chargaff, 1959). Samples of 1.0 or 1.5 ml. were withdrawn at different times from the aerated, phage-infected cultures and precipitated in 5.0 ml. and 7.5 ml., respectively. of 6%, cold trichloroacetic acid. The precipitates were collected on membrane or glass fibre filters (Gelman, Metricel GA-6 and Whatman GF/C, respectively). Radioactivity was measured in a Packard 3375 scintillation counter after immersing the dried filter in naphthalene-dioxane scintillation fluid (Bray, 1960). When 14C and 3H were measured in the same sample, spectrometer settings were used which gave less than 0.5% of the tritium activity in the i4C channel. Since the aH/14C ratio of the incorporated activities usually varied between 05 to 8.0, this tritium overlap was ignored. About 3076 of the i4C counts appeared in the 3H channel and appropriate corrections were made.
342
0. (f) Measurement
SKC)LD
of cellular
ti-fhorouracd
nucleotido
pools
The incorporation of 3H-labelled-5-fluorouracil into the nucleoside mono-, di- and triphosphate pools of phage-infected cells was determined after nucleotide separation by high voltage electrophoresis of perchloric acid extracts. At different times after infection, 2-m]. samples were withdrawn from the infected cultures containing 3H-labelled FU, and filtered on a membrane filter (Gelman, Metricel GA-6). The cells were rapidly washed on the filter wit,h 2 x 1 ml. of mmeral salts medium without glucose, and finally suspended in 1 ml. of cold 0.4 M-perchloric acid. After centrifugation the supernatant fraction was neutralized with 1 M-KOH and the perchlorate precipitate centrifuged off. Samples of this supernatant fraction were subjected to electrophoresis on Whatman 3 MM paper in a 0.05 M-citrate buffer, pH 4.6, for 2 hr at 60 v/cm. Samples of UMP, UDP and UTP could be used as references, since initial test runs showed these nucleotides to move identically with the corresponding FU-nucleotides at the pH used. The nucleotide spots were localized by ultraviolet light, cut out, the paper discs immersed in 20 ml. of naphthalene-dioxanc scintillation fluid (Bray, 1960), and counted in a Packard scintillation counter.
3. Results (a) Inhibition
of bacteriophuge production by 5-jluorouracil at high temperature
The effect of FU on T4 phage production in E. coli B at different temperatures is demonstrated in Table 1. In the presence of 10 pg FU /ml. and at an elevated temperature (42”C), phage production is seen to be completely abolished. The same concentration of FU does, however, allow phage production at lower temperatures. The burst size is thus decreased by only 30% at 26°C. The heat-sensitizing effect of FU on phage development is reversible, which is shown in Figure 1. It can be seen that phage production in the uracil-requiring mut,ant OK302 in the presence of FU recovers if the temperature is shifted from high (41°C) to low (3O”C), and with increasing efficiency the earlier the shift is made. The two lower curves of Figure 1 are controls, without temperature shift and run at 41 and 43”C, respectively. Some production of phage can be observed late in the infection period at 41”C, whereas the inhibition is complete at 43°C. That the high temperature per se did not affect phage development in OK302 is shown in Figure 2, which demonstrates intracellular phage production at 41G”C TABLE
1
Temperature sensitivity of bacteriophage production in the presence of 5-jluorouracil Temperature
42 35 30 26
(“C)
Burst
(% of control)
0 20 35 70
Cells, E. wli B, were grown and infected with T4 as described. The adsorption mixture containing 10 pg FU/ml.; 20 pg thymidine/ml. and cells (1.2 x 10’O/ml.) was warmed to the experimental temperature before addition of phage. After phage adsorption for 3 min without aeration, the infected complexes were diluted lo-fold into aerated mineral salts medium of the experimental temperature and containing 10 pg FU/ml. and 20 pg thymidine/ml. Lysis was induced with chloroform after 60 min of incubation. The results are given as percentage of control burst size, obtained in t,he absence of FU at all temperatures (130 to 140 per infective centre).
REGULATION
OF T4 RNA
343
SYNTHESIS
4041° c +30°
c
30-
//Ii
a,&(,&
IO
20
30
40
50
60
70
80
Time (mln)
FIG. 1. Reversibility of FU effect by temperature shift. Cells, OK302, (uracil-) were grown and infected as described. The adsorption mixture which contained 10 pg FU/ml. and 20 pg thymidinelml. was warmed to 41°C before phage infection. The infected cells were diluted lo-fold into mineral salts medium at 41°C containing 10 pg FU/ml. and 20 pg thymidine/ml. Shifts to 30°C were performed at the indicated times. At the different points samples were withdrawn and lysed with chloroform. Results are given as number of phage produced per bacterium. The two lower curves are from experiments without temperature shift. --x--x--,-A--A--, and -O--O--, 41°C; --n--n--, 43’C.
(lysis induced with chloroform). The formation of mature phage was observed to start 10 to 12 minutes after infection and proceeded almost linearly until 40 minutes after infection. (b) Rescue by uridine of bacteriophage production inhibited by 5-jluorouracil at a high temperature Not only shift in temperature but also the addition of uricline was shown to reverse the inhibition of phage production by FU at a high temperature. This is most simply explained as a chasing out of FU by uridine from the metabolically labile phage mRNA. The phenomenon is demonstrated in Figure 3, where the lowest curve shows an almost complete inhibition of phage production at 41.5% in the presence of FU 10 pg/ml. When uridine was added to a final concentration of 150 pg/ml. at seven minutes after infection, phage production was observed to approach the values of the control without FU. Addition of uridine at later times after infection gave a gradually diminishing response. It thus seemed as if FU was chased from phage RNA wit)h decreasing efficiency at later times after infection.
I 0
IO
20
30
40 50 Time (mid
60
70
8C
FIG. 2. Phage production at high temperature in the uracil-requiring E. co& B strain OK302. Phage infection was performed as described. The temperature W&S 41.5”C, and the adsorption mixture containing 10 pg uridine/ml. was warmed before the addition of phage. After adsorption the infected cells were diluted IO-fold into mineral salts medium at 41.5”C containing 10 pg uridine/ml. At the indicated times, samples were withdrawn, lysed with chloroform and assayed for phage. The results are given as number of phages produced per ml. of aerated growth culture.
160-
60-
0
1
60
80
100
Time (min)
FIG. 3. Uridine rescue of phage production inhibited by FU at a high temperature. The experiments were performed as described in the legend to Fig. 1 with strain OK302 as host, and at 41.5’C. The upper curve represents a control without FU but with 10 pg uridine/ml. At the indicated times, uridine was added to a final concentration of 150 pg/ml. Results are given as phages produced per input bacterium.
REGULATION
(c) Inhibition
of bacteriophage
OF T4 RNA
DNA
345
SYNTHESIS
synthesis by 5-jluorouracil
at high temperature
The inhibiting effect of FU on phage production at a high temperature could br explained by its interfering witch early protein formation via its incorporation ink, early mRNA. This interpretation is supported by the experiment shown in Figure 4. Phage-specific DNA synthesis, measured as the cumulative incorporation of radioactive thymidine into acid-insoluble product, was seen to be almost completely inhibited by the presence of FU at 41°C. The inhibition could, however, be reversed b> shifting the temperature from 41 to 30°C at seven and nine minutes, respectively, after infection. The rescue of DNA synthesis was significantly more efficient with temperature shift at seven minutes than at nine minut.es. The FU-inhibition of phage DNA synthesis at a high temperature is not a conscquencr of a general toxic effect of FU on the cells. This is demonstrated in Figure 5(a), I
I
I
I
I
8000 -
2000 -
Time (min) Fm. 4. Inhibition
of phage DIiA
synthesis by FU at high temperature and its reversion by temperature shift. Cells (OK302) were grown and infected as described. Synthesis of DNA was measured by the cumulative incorporation of [sH]thymidine into acid-insoluble product as described. The phage was allowed to adsorb for 3 min at 41.5% and the infected cells were then diluted lo-fold into mineral salts medium at 41.5”C containing [eH]thymidine 20 pg/ml; 1 PC/ml. and FU 10 pg/ml or uridine 10 pg/ml. (control). The values along the ordinate are expressed as counts per min. incorporated per 1.5ml. sample. Phage infection at time zero. -O-O-, +- FU, no temperature shift; -O-O-, + FU, temperature shift to 30°C at 9 min; -A-A--, + FU, temperature shift to 30°C at 7 min; -xx--, control with uridine, temperature shift to 30°C at 13 min.
3ir
I IO
I 20
30
40
I 50
60
Fm. 5. Absence of toxic effects by FU on host cells. (a) Cells (strain OK302) were grown, harvested, washed and resuspended as described. The experiment was started by diluting the uninfected cells lo-fold into mineral salts medium warmed to 42’C containing 20 pg [3H]thymidine/ml., 1 pc/ml., and 10 pg FU/ml. or 10 pg uridine/ml. (control). Values along the ordinate are expressed as counts per min per l&ml. sample. - x - X -, -+ 10 pg FU/ml.; -O-O-, control with 10 pg uridine/ml. (b) Cells (OK302) were prepared as in (a). The experiment was started by diluting the uninfected cells IO-fold into mineral salts medium containing 20 pg thymidine/ml. and 10 pg FU/ml. or 10 pg uridine/ml. (control). After incubation at 41°C for 20 min the cells were cooled, concentrated lo-fold by centrifugation and infected by phage. After adsorption for 3 min at 41°C the infected cells were diluted lo-fold into mineral salts medium at 41°C containing 20 pg [3H]thymidine/ml., 1 PC/ml. and 10 pg uridine/ml. but no FU. Values along the ordinate are expressed as counts per min per 1.5ml. sample. Phage infection at time zero. - x - X-, cells preincubated + 10 rg FU/ml.; -O-O-, control cells preincubated with 10 pg uridine/ml.
where the DNA synthesis in uninfected cells of the uracil-requiring E. coli B strain OK302 is seen to be completely unimpaired by the presence of FU at 42°C. That FU really entered the RNA under these conditions is shown below (Fig. lO( b)). The decrease in C3H]thymidine incorporation observed at 30 to 40 minutes after the addition of labelled precursor is probably explained by the enzymic degradation of thymidine to thymine, which is a very poor precursor for DNA synthesis in uninfected but not in T-even phage infected cells (Kemmen & Strand, 1967; Munch-Petersen & Vilstrup, 1969, Abstracts, Fed. EUTO~.Biochem. Sot., no. 873, &lad&d). Another experiment to check for the possible toxic effect of FU on the host cells is shown in Figure 5(b). In this c&se the uninfected cells were pretreated with FU (10 pg/ml.) at 41°C for 20 minutes, centrifuged, washed and finally resuspended in the absence of FU and infected by phage at 41°C. The FU-pretreated cells were observed to support phage DNA synthesis just as well as the control.
REGULATION
OF
T4 RNA
347
SYNTHESIS
The effect of FU on protein and DNA synthesis, respectively, was furthermore directly compared in phage-infected cells. In the experiment presented in Figure 6, phage DNA and phage protein synthesis were measured simultaneously in cells infected at 41.5”C and in the presence of FU. Rate and extent of protein synthesis is seen I
150 -
I
I
I
3H activity
A-A-A
A-A-A-
IO
(a)
Time (mm)
30
20
40
(b)
Fro. 6. Effect of FU on protein synthesis at a high temperature. The experiment was performed at 41.5”C as described in the legend to Fig. 4. The infected cells (OK302) were diluted lo-fold into mineral salts medium containing 20 pg [3H]thymidine/ml., 1 &ml.; 10 pg [W]leucine/ml., 0.05 PC/ml. and 10 pg FU/ml. or 10 pg uridine/ml. (control). In one control experiment the dilution medium also contained 20 pg chloramphenicol/ml. The 3H- and 14C-radioaotivities were separated in the scintillation counter as desoribed in the Materials and Methods section. The values along the ordinate are expressed as counts per min incorporated per 1 ml-sample of sH and W activity, respectively. Phage infection at time zero. control with 10 pg uridine/ml.; -s--O-, + 10 pg FU/ml.; -A-A-, control -x-x-, with 10 pg uridine/ml. and 20 pg chloramphenicol/ml.
to be indistinguishable from those of the control with uridine instead of FU. Phage DNA synthesis was markedly inhibited by FU, although not completely, in this experiment (of. curve obtained in the presence of chloramphenicol). The quantity of proteins produced in the presence of FU was thus unimpaired. Their quality on the other hand seemed to be changed as reflected by the inability of the early enzymes produced, to support a normal phage DNA synthesis. (d) Rescue by uridine of bacteriophage DNA synthesis inhibited by 5-$uorouracil at a high temperature The pronounced inhibitory effect of FU on phage DNA synthesis at a high temperature could be reversed by the addition of uridine. As demonstrated in Figure 7 this reversion became, however, less pronounced, the later uridine was added after infection. Thus uridine addition at seven minutes gave a DNA synthesis which was about
348
0.
SKC)bD
Fra. 7. Uridine rescue of phage DNA synt,hesis inhibited by FLJ at a high kmperature. The experiment was performed as described in the legend to Pig. 4 and at 41’C. The infecked C& (strain 0X309) were diluted lo-fold into mineral salts medium containing 20 pg [W]thymidine/ml., 1 hq’rnr. and 10 pg l?U/ml. OP 10 ~6 uridinejml. (control). At the times indicated on each curve, uridina was added to the infected cultures to a final concentration of 150 pg,knl. The values along the ordinate are expressed as counts per min incorporated per 1.5.ml. ssmpls. Pbage infection at time zero. -o-O--, + FU, 160 pg uridine/ml. added at - x - x -, Control wit)h lO,ug uridine/ml.; 7min; -A--&--, + FU, 150 @ uridinc + FU, 150 ~IF;nridine/ml. added at 9 min; --n--A--, {ml. added at 18 min; --+-a--, + EZT, 150 pg uridine/ml. added at 24 min.
that of the control. Uridinc addition at nine minutes and 22 minutes gave a synthesis of 14 and ‘I%, respectively. The reversion of IMA synthesis inhibition thus foIlowed a pattern similar to that for uridine rescue of EJ-inhibited phage production shown jn Figure 3. The discrepancy between appa,rent efficiency of uridine rescue of phage production (Fig. 3) and of DNA synthesis (Fig, 7) is probably explained by tke fact that cumulative incorporation of ~3H]thymidine measured total DNA synthesis, e.g. alao Ghe formation of DNA which will never be incorporated into pbage. The relatively large phage production even after relatively long periods of FU inhibition (Fig. 3) would then represent an eficient utilization of the small amounts of DNA formed during FU inhibition (cf. Fig. 6). The measurement of total DNA synthesis
SO$/, of
REGULATION
OF T4 RNA
SYNTHESIS
340
would then, according to this argument, more closely reflect the total capacity of the early enzymes formed. The rapidly diminishing efficiency of uridine rescue of FUinhibited DNA synthesis is interpreted to reflect a decreasing synthesis of early mRNA. Uridine could chase out FU from early mRNA only as long as the latter is actively turning over. A diminishing early mRNA synthesis would support the synthesis of decreasing amounts of early enzymes, and successively of DNA. (e) Early mRNA synthesis in gene 33 and gene 55 TQ-natiants, resected us uridine rescue of 5-$uorouracil-inhibited DNA fornaation The amber mutants amN134 (gene 33) and amBL292 (gene 55) are known as maturation deficient, i.e. they will perform nothing or very little of the late functions, although they are capable of DNA synthesis (Bolle et al., 19686). Since these mutants seem to have a defect in the normal change from early to late events during phage development,, the kinetics of their early mRNA syntjhesis was studied wit*h the method described in the previous section. They were thus tested for uridine reversion of the
Time
FIG. The control uridine
(min)
8. Uridine rescue of mutant amBL292 DNA synthesis inhibited by FU at a high temperature. experimental procedure was identical to that described in the legend to Pig. 7. - x - x --, with 10 pg uridine/ml.; -A-A--, -I- FU, uridine added at 14.5 min; -A-A-, + FIT, added at 22 min; -O--c--, + FU, uridine added at 25 min.
0.
350
SKC)LD
FU inhibition of their DNA synthesis. With the mutant amBL292 the decrease in reversion efficiency with time of uridine addition was much less pronounced t’han with wild type as shown in Figure 8 (cf. Fig. 7). Addition of uridine at 22 to 25 minutes after infection in the presence of FU at 41°C could thus rescue substantial amounts of mutant DNA synthesis. A very similar behaviour was found for the mutant anziV134 (gene 33) (data not shown). Figure 9 shows a comparison of the uridine rescue of
I 0
I IO
I
J
20 Time (min)
FIQ. 9. Comparison of uridine rescue of FU-inhibited DNA synthesis in anaBL292 and T4 infection. Replot of data from Figs 7 and 8. Incorporation of [aH]thymidine between 40 and 60 min after mutant and wild-type phage infection, respectively, in FU inhibited cultures. Values are expressed as percentages of the corresponding values for controls without FU and plotted Z)WSW time of uridine addition. ---AA-, amBL292 infection; - x - x -, T4 + infection.
BU-inhibited DNA synthesis after amBL292 (gene 55) and wild type T4 infection, respectively. The cumulative 13H]thymidine incorporation between 40 and 60 minutes. after infection in B-m-inhibited cultures is plotted versus time of uridine addition. The late time period for DNA determination was chosen to avoid interference with the late addition times of uridine. A clear difference between mutant and wild type infection can be seen. When uridine was added at, for example, 14.5 minutes after infection 96% of mutant DNA synthesis was rescued in contrast to only 5 to 8% of that of the wildtype. (f) Incorporation
and chase of 5-$uorouracil in bacterio@uzge-infected non-infected
and
cells
A direct demonstration of the chasing out of FU from phage mRNA with uridine is shown in Figure IO(a). The radioactivity incorporated from l*C-labelled FU into acidinsoluble RNA is seen to disappear rapidly after the addition of uridine. The chase is not complete, however, which is interpreted as a reutilization of labelled FU nucleotides for the synthesis of late phage mRNA. A chase performed at 15 minutes gave a similar pattern (data not shown). In one experiment with uridine chase at seven minutes, phage DNA synthesis was measured simultaneously. It can be seen, that the initially inhibited DNA synthesis was released about 11 minutes after uridine addition which corresponds to thr time period Mn-ten infection and start’ of DNA synthesis in
REGULATION
0
IO
’
’
20
30
OF T4 RNA
40
0
351
SYNTHESIS
IO
20
30
40
Time (mln)
(a) (b) 10. Chasing of FU from RNA of phage-infected and uninfected cells (OK302). (a) The experiment was performed as described in the legend to Fig. 4 and at 41°C. The infected cells (strain OK302) were diluted lo-fold into mineral salts medium containing 20 pg thymidine/ml. FU/ml., 0.05 pc/ml. At the (3H-labelled, 1 &ml., in one experiment) and 10 pg l*C-labelled indicated times uridine was added to the infected cultures to a final concentration of 150 pg/ml. 14C- and 3H-activities were separated in the scintillation counter. The values along the ordinates are expressed as counts per min incorporated per l-ml. sample. Phage infection at time zero; - x - x -, no uridine chase; -O-O-, uridine added at 7 min; -n-n-, uridine added at 12 min; --A--A--, DNA synthesis ([3H]thymidine incorporation) in the experiment with uridine addition at 7 min. (b) Bacterial cells (OK302) were prepared as described. The experiment was started by diluting the cells IO-fold into mineral salts medium at 30°C containing 20 pg thymidine/ml. and 10 pg 3H-labelled FU/ml., 1 PC/ml. The values along the ordinate are expressed as counts per min incorporated per l-ml. sample. - X-X -, no uridine chase; -O-O--, uridine added to 150 pg/ml. at 8.5 min. FIG.
normal infection (cf. Fig. 7). Phage-infected cells incorporate little l*C-labelled FU int.0 acid-insoluble product later than ten minutes after infection (Fig. 10(a), upper curve). In contrast, uninfected cells (Fig. 10(b)), which synthesize stable RNA, continue to incorporate large amounts of 3H-labelled FU for the studied time period of 40 minutes. In concurrence with this observation the radioactivity incorporated from l*C-labelled FU into the RNA of uninfected cells could not be chased with uridine (Fig. 10(b)).
(g) Rate of early mRNA synthesis at different times after bacteriophage infection In order to obtain a more direct evaluation of the changing intensity in early mRNA formation with time after infection, the rates of early mRNA synthesis were mea.sured by determining the short-time (1 min) incorporation of l*C-labelled FU into acidinsoluble product after infection with phage and’42 (gene 1). This mutant is deficient in dHMP-kinase and cannot induce any formation of DNA in E. coli B. Since there is 24
352
0.
SKi)LD
Time (mid FIQ. 11. Rates of RNA synthesis at different times after infection with mutants and wild type of T4. Cells of E. coli B were grown and infected as described, but FU was not added to the adsorption mixture. After adsorption for 3 min at 37°C the infected cells were diluted lo-fold into mineral salts medium at 37°C containing 20 pg thymidine/ml. At the different times sH-labelled FU was added to 10 pg/ml. and 1 &nl. Samples of 1 ml. were withdrawn and precipitated with trichloroacetic acid after 1 min. The values are plotted as percentages of the 1-min incorporation values for eH-labelled FU addition at 3 min. These values were in counts per min per 1 ml.-sample: 207 for amC42, 135 for anaC42 x arnBL292 and 126 for T4+.
no DNA synthesis in amC42-infected E. coli B, there is no formation of late mRNA (Bolle et al., 196%). This means that any incorporation of l*C-labelled FU into acidinsoluble product after infection must represent early mRNA. In Figure 11 some experiments are demonstrated, in which the incorporation of 14C-labelled FU added at different time points after infection is measured during one minute periods. The data are plotted as percentages of the incorporation obtained at three minutes, when the incorporation was maximal. A comparison of the diagrams for amC42 (top part) and T4+ (bottom part) in Figure 11 shows that the short-time incorporat,ion of l*C-labelled FU diminishes rapidly with time of pulse addition in cells infected with the mutant. Pulses given at 15 and 20 minutes, respectively, hardly give any observable incorporation with the mutant, which indicates a rapidly diminishing synthesis of early mRNA. The incorporation with T4 at 20 minutes is about 35% of that at three minutes, representing mainly late mRNA synthesis. In Figure 11 is also shown an
REGULATION
OF T4 RNA
353
SYNTHESIS
experiment with a double mutant amC42 x amBL292 (gene I x gene 55). It can be seen that the presence of both mutations in the same genome prolonged the time period of early mRNA synthesis in the mutant-infected cells. The pulse incorporations at 15 and 20 minutes were 58 and 2Oo/oof that at three minutes, respectively, and thus substantially higher than the corresponding values for the gene 1 single mutant. These results imply that gene 55 is in some way connected to the regulation of early mRNA synthesis. The low pulse incorporation of 3H-labelled FU into phage RNA at later times with the amC42 mutant could be due to a diminishing uptake of the labelled base into the nucleotide pool. To rule out this possibility, labelled 5-FU was added to amC42-infected cells at 3 and 12 minutes, respectively, and its incorporation into nucleoside mono-, di-and triphosphate pools was determined by analysis of extracts with high voltage electrophoresis. The results are presented in Table 2. It can be seen that the general TABLE
2
Uptake of 3H-labelled PU into the nucleotide pools of amC42- and T4+-infected cells 3H-labelled Time (min) 6 11 23
FUMP 100 245 1112
3H-labelled
FU added at 3 min amC42 FUDP 238 473 525
FUTP 415 1012 1780
T4 FUTP 560 1462 1670
Tim0 (mm) 18 23 32
FUMP 275 415 320
FU added at 12 min amC42 FUDP 388 440 507
FUTP 762 810 1330
T4 FUTP 830 1430 2090
E. coli B cells were grown and infected with phage as described, but FU was not added to the adsorption mixture. After phage adsorption for 3 min at 37°C the infected cells were diluted 6.4fold into mineral salts medium at 37% containing 20 pg thymidine/ml. and 9.5 ng FU/ml. At 3 min and 12 min, respectively, after infection, 3H-labeled FU was added to 7.8 pc/ml. At the indicated times after infection 2-ml. samples were withdrawn and analysed as described in the Materials and Methods section under (f). The values are expressed as counts per min per 3.5 x lo7 cells.
incorporation pattern of 3H-labelled FU into the different nucleotide pools is not changed dramatically when the labelled precursor is added at 12 minutes after infection as compared to addition at three minutes. Formation of FUTP, which is the immediate precursor of FU-containing RNA, is seen to continue during the period 12 to 32 minutes after infection. The uptake of FU into the nucleotide pools of amC42infected cells is thus not restricted at late times after infection. For comparison the 3H-labelled FU uptake into the FUTP pools of T4+-infected cells is also shown in Table 2.
4. Discussion In these experiments I have investigated the regulation of early mRNA formation in T4 phage-infected E. coli B cells. Two different approaches were used. One was functionally to “label” newly formed mRNA specifically involved in phage-specific DNL4 synthesis. The other was to measure rates of early mRNA synthesis by radioactive pulse labelling. Gros & Naono (1961) showed that E. coli cells grown in the presence of FU produced an alkaline phosphatase which was much less thermostable than the enzyme formed in
354
0.
SKOLD
the absence of the analogue. They concluded that FU was incorporated into the mRNA corresponding to alkaline phosphatase. A similar effect was observed in the present experiments, where the presence of FU at a high temperature was seen to abolish phage production and more specifically phage DNA synthesis. The known inhibitory effect of the FU-deoxynucleotide on thymidylate synthetase (Cohen et al., 1958) was neutralized by the addition of thymidine in all experiments. The observation that both DNA formation and phage production could be restored by shifting to a lower temperature, indicates a FU-induced heat-lability in one or several of the enzymes responsible for phage DNA synthesis. This interpretation is further supported by the finding that bulk synthesis of phage proteins is unimpaired under conditions when FU severely restricts phage DNA formation. That the effect of FU is directed towards enzymes being synthesized is furthermore indicated by the insensitivity of host bacterial DNA synthesis to the presence of FU at a high temperature. Both phage-infected and uninfected bacteria readily took up FU into RNA. I conclude that the inhibitory effect of FU on phage DNA synthesis is a consequence of its incorporation into phage mRNA, where it introduces coding errors, which are finally expressed as a decreased thermostability of early phage enzymes. Inhibition by FU of both phage production and phage DNA synthesis at high temperature was reversed by the addition of uridine, which efficiently enters labile RNA as uracil. This is interpreted as a metabolic dilution of FU from mRNA to allow the formation of mRNA without coding errors and consequently of normal early enzymes. The effect on phage DNA synthesis of this metabolic chase of FU from mRNA could however, be observed only as long as active synthesis of that mRNA fraction which is responsible for the formation of DNA-synthesizing enzymes proceeds. The experiments demonstrated a decrease in the efficiency of uridine rescue of both phage production and phage DNA synthesis with time after infection. This indicates a gradual decrease in the synthesis of mRNA coding for early enzymes, because the later uridine is added, the less early enzymes in heat-stable form would then eventually become available for DNA synthesis. The pattern of uridine rescue of DNA synthesis was quite different after infection with mutants of phage T4 carrying lesions in either one of genes 33 and 55. In these cases, DNA synthesis at high temperature could be rescued from FU-inhibition at much later times than in wild-type infection, indicating a much slower decrease in early mRNA synthesis. This is in accordance with the results of Hosoda & Levinthal (1968), who could show a delay in the time when early protein synthesis is normally stopped after infection with genes 33 and 55 mutants, respectively. Since the uridine rescue is mediated by the early mRNA synthesis, I conclude that the experiments indicate the presence of a transcriptional regulation mechanism which terminates the synthesis of early, DNA-synthesizing enzymes, and that this mechanism is dependent on an undisturbed function of genes 33 and 55, respectively. The interpretation of these experiments relies on the efficiency of the uridine chase of FU from mRNA at different times after infection. For this reason the incorporation kinetics of FU and of uridine into phage mRNA was given special attention. FU into phage mRNA at the high The cumulative incorporation of l*C-labelled temperature was essentially finished at about ten minutes after infection, at which time a plateau was reached (Fig. 10(a)). Since very little (Weiss, Hsu, Foft & Soherberg, 1968) stable RNA is formed after phage infection, this plateau should represent
REGULATION
OF T4 RNA
SYNTHESIS
355
an equilibrium between uptake of 14C-labelled FU into mRNA and mRNA degradation. According to Nierlich (1967) the uptake of a radioactive RNA precursor would take place only as long as net synthesis of RNA proceeds. The initial branch of the 14Clabelled FU incorporation curve (Fig. 10(a)) should then represent an expansion of the pool of phage mRNA. That an increase in the cellular contents of labile mRNA does occur in the early period after infection is supported by the findings of Landy & Spiegelman (1968) and Kleppe & Nygaard (1969). The plateau of the incorporation curve has a slight slope of increase, which, however, extends far beyond the time (10 to 12 min) when early mRNA formation according to the uridine rescue experiments would be essentially finished. The RNA synthesis corresponding to the latter portions of this plateau would then consist of late mRNA synthesis. It is, however, known that no late mRNA synthesis could be initiated without preceding phage DNA synthesis (Bolle et al., 19683), which in this case ought to be inhibited by FU at the high temperature. The synthesis of late mRNA is explained here as a result of an incomplete FU-inhibition of phage DNA formation (could be seen in Fig. 6). It is known that a small amount of DNA synthesis, for a short period during phage development, is sufficient to allow late mRNA to be formed (Bolle et al., 19686). This interpretation is supported by the observation that FU-label could be chased from phage RNA as late as 12 to 15 minutes after infection. At these late times, the uptake of RN-4 precursors would then be effected through the slow expansion of the late mRNA pool and possibly also by expansion of the nucleotide pools (discussed below). I conclude that FU can be efficiently chased from phage RNA by uridine during the initial 15 minutes of the infection period, but that only chases earlier than 9 to 12 minutes can rescue the synthesis of early enzymes involved in phage DNA synthesis. The determinations of total phage RNA synthesis, by short-time labelling at different times after infection, also indicated the presence of a transcriptional regulation mechanism which switches off early mRNA synthesis. The rates of total RNA formation thus decreased rapidly with time after infection with mutant an&‘42 (gene l), which can induce only early mRNA synthesis. The decrease in early mRNA synthesis was, however, much slower in a double mutant containing a mutation also in gene 55. These results support the conclusion that gene 55 is involved in the transcriptional regulation of early mRNA formation. The validity of the short-time labelling experiments depends on the ability of the labelled precursor to enter the nucleotide pools. According to Nierlich (196’7), labelled precursor would be taken up into the nucleotide pools only as long as there is a net utilization of nucleotides for RNA synthesis under conditions where nucleotide pools do not expand. In the present experiments where presumably no net synthesis of RNA occurs this would mean that 14C-labelled FU would enter the nucleotide pools only as long as the pool of labile phage mRNA expands. If this expansion ended early (cf. discussion above of experiment in Fig. 10(a)) the interpretation given above would be invalidated, It was, however, shown by the direct analysis of nucleotide labelling, that the uptake of label into nucleotides continued at, a substantial rate as late as 23 to 32 minutes after infection, under conditions when only early mRNA formation takes place. This period is far beyond the time when early mRNA synthesis is shut off as discussed above. These results could be explained by an expansion of the nucleotide pools, which is known to occur in bacteria after the arrest of net RNA synthesis (Edlin & Stent, 1969).
356
0. SKOLD
This research was supported by a grant from the Swedish Medical Research Council. REFERENCES Belle, A., Epstein, R. H., Salser, W. & Geiduschek, Bolle, A., Epstein, R. H., Salser, W. & Geiduschek, Bray, G. A. (1960). Analyt. Biochem. 1, 279. Cohen, S. S. (1948). J. BioZ. Chem. 174, 281. Cohen, S. S. (1963). Ann. Rev. Biochem. 32, 83. Cohen, S. S. (1968). Virus-induced Enzymes, New Cohen, S. S., Flaks, J. G., Barner, H. D., Loeb, M. Acad.
Sci.,
E. P. (1968a). J. Mol. Biol. 31, 325. E. P. (1968b). J. Mol. Biol. 33, 339.
York: Columbia University Press. R. & Licht,enstein, J. (1958). Proc. Nat.
Wash. 44, 1004.
Edgar, R. S. & Wood, W. B. (1966). Proc. Nut. Au&. Sci., Wash. 55, 498. Edlin, G. & Stent, G. S. (1969). Proc. Nut. Acad. Sci., Wash. 62, 475. Gros, F. & Naono, S. (1961). In Protein Biosynthesis, ed. by R. J. C. Harris, p. 195, New York : Academic Press. Hall, B. D., Nygaard, A. P. & Green, M. H. (1964). J. Mol. BioZ. 9, 143. Horowitz, J. & Chargaff, E. (1959). Nature, 184, 1213. Hosoda, J. 8: Levinthal, C. (1968). Viirology, 34, 709. Kammen, H. 0. & Strand, M. (1967). J. BioZ. Chem. 242, 1854. KarlstrGm, 0. (1968). J. Bact. 95, 1069. Khesin, R. B., Gorlenko, Zh.M., Shemyakin, M. F., Bass, I. A. & Prozorov, A. A. (1963). Biokhimya, 28, 1070. King, J. & Wood, W. 13. (1969). J. Mol. BioZ. 39, 583. Kleppe, R. K. & Nygaard, A. P. (1969). Biochim. biophys. Actu, 190, 202. Kornberg, A., Zimmerman, S. B., Kornberg, S. R. & Josse, J. (1959). PTOC. Nat. Acad. Sci.,
Wash. 45, 772.
Landy, A. & Spiegelman, S. (1968). Biochemistry, 7, 585. Nierlich, D. P. (1967). Science, 158, 1186. Nomura, M., Okamoto, K. & Asano, K. (1962). J. Mol. BioZ. 4, 376. Nomura, M., Witten, C., Mantei, N. & Echols, H. (1966). J. Mol. BioZ. 17, 273. Salser, W., Belle, A. & Epstein, R. (1970). J. Mol. BioZ. 49, 271. SkGld, 0. & Buchanan, J. M. (1964). Proc. Nat. Acad. Sci., Wash. 51, 553. Weiss, S. B., Hsu, W., Foft, J. W. & Scherberg, N. H. (1968). Proc. Nat. Acad. Sci., Wash. 61, 114.
Wiberg, J. S. (1966). Proc. Nut. Acad. Sci., Wash. 55, 614. Wiberg, J. S., Dirksen, M., Epstein, R. H., Luria, S. E. & Buchanan, Nat. Acad. Sci., Wash. 48, 293.
J. M. (1962). Proc.
Regulation of Early RNA Synthesis in Bacteriophage T&infected Escherichia coli Cells OLA SK~LD Department
of Microbiology,
Faculty
of Pharmacy
University of Uppsala, Xweden (Received 22 April
1970, and in revised form 9 July 1970)
The regulation of synthesis was examined for those early mRNA species in bacteriophage T4 infection which are responsible for the formation of early enzymes involved in DNA synthesis. Evidence was found for a transcriptional regulation mechanism, connected to the function of phage genes 33 and 55. Early mRNA formation was observed to be sharply turned off at about the time of normal initiation of DNA synthesis. This distinct shut-off was abolished if the infecting phage contained a lesion in gene 33 or 55. The determination of synthesis of those early mRNA species responsible for phage DNA formation was based on the observation that the presence of 5-fluorouraoil made the formation of phage DNA heat-sensitive. This was interpreted as a consequence of fluorouracil incorporation into early mRNA, where it introduces coding errors, which in turn cause heat sensitivity of the phage-specific, DNAforming enzymes. The fluorouracil inhibition of phage DNA synthesis could be reversed either by a shift-down in temperature or by the addition of a high concentration of uridine. In the latter case the efficiency of rescue decreased rapidly with time of uridine addition after infection. This is thought to reflect the formation of those mRNA species which are responsible for the synthesis of DNAforming enzymes. Uridine could chase out fluorouracil from early mRNA and allow synthesis of error-free messenger and subsequently of heat-stable early enzymes, but only as long as the synthesis of these particular mRNA species proceeded. The turn-off of early mRNA synthesis and its dependence on an undisturbed gene 55 function could also be demonstrated by short-time labelling of total RNA, which formed after infection with early mutants producing only early mRNA. In this case the uptake of labelled precursor into nucleotide pools at different times after infection was checked by electrophoretic analysis.
1. Iutroduction For many years it has been well established, that when a T-even phage, such as T4, infects a sensitive Escherichia coli cell, host macromolecular synthesis stops immediately and RNA, DNA and proteins controlled by the viral genome start to be synthesized (Cohen, 1948; Nomura, Okamoto & Asano, 1962; Nomura, Witten, Mantei & Echols, 1966). In the early period after infection many phage-specific proteins are synthesized which will not be incorporated in the mature phage. Many of these early proteins have been demonstrated to be enzymes involved in the biosynthesis of phage DNA (Cohen, 1963, 1968). Most of the proteins formed during the later period after infection become 339
340
0.
SKOLI)
part of the mature phage as structural proteins. The late pathlrays of assembly ar(s under investigation (Edgar & Wood, 1966 ; King & Wood, 1969). Results of Hosoda & Levinthal (1968) show that this simple classification of phage proteins into early and late is not adequate. By protein labelling, disc elcctrophoresis and autoradiography they have distinguished a group of phage proteins, the synthesis of which begins much more rapidly after infection t’han does synthesis of most of the early proteins. The class of early phage proteins is thus not controlled as a single homogeneous group, but synthesis of its members is both initiated and shut ofI’ at various times during the early part of the infect,ion process. The best-characterized of the early proteins arc those involved in phage DNA synthesis (Cohen, 1968) and for this group of proteins, there seems to exist a co-ordinate regulation mechanism, which shuts off net synthesis of enzymes at a definite time after infection (Kornberg, Zimmerman, Kornberg & Jossc, 1959; Wiberg, Dirksen, Epstein, Luria & Buchanan, 1962). The general features of the temporal sequence of phage protein synthesis are reflected in the changing pattern of hybridizable RNA, pulse-labelling in phage-infected cells (Khesin, Gorlenko, Shemyakin, Bass & Prozorov, 1963; Hall, Nygaard & Green, 1964). Furthermore Belle, Epstein, Salser & Geiduschek (1968a) could show by the refined technique of RNA-DNA hybridization-competition, that at 20 minutes after infection, at 30°C “late” RNA species were several hundredfold more abundant than at five minutes after infection, and in a later work (Salser, Bolle & Epstein, 1970) they showed that “true-early” RNA decreased severalfold in level between 5 and 20 minutes after infection. They concluded that these observations reflected changing rates of transcription. The experiments described in this paper aim at studying the regulation of synthesis, specifically of those mRNA species responsible for the formation of early enzymes involved in phage-specific DNA synthesis. The experimental approach was based on the observation, that the presence of 5-fluorouracil in the medium made phage production, and more specifically phage DNA synthesis heat-sensitive. Both DNA synthesis and phagc production could, however, be rescued at the high temperature by the addition of uridine. The efficiency of this uridine rescue rapidly diminished wit,h time after infection. The inhibition of phagc DNA synthesis by FUt (in the presence of thymidine) was explained by its incorporation into early mRNA, thus inducing coding errors and making phage enzymes involved in DNA synthesis heat-sensitive. The rescue from the FU inhibition by uridine would then take place by the chasing out of FU, but only as long as active synthesis of early mRNA proceeded. The observed decrease in rescue efficiency with time after infection is interpreted to reflect a normal regulation of early RNA synthesis. Mut,ations in genes 33 and 55 were found to interfere with this regulation.
2. Materials and Methods (a) Bacteria
and Bacteriophage
E. coli B and a uraoil-requiring mutant of it (OK 302, Karlstrom, 1968) was used. Wild type bacteriophage T4D and mutant rcmC42 (gene 1, hydroxymethyl deoxycytidylate kinase) was obtained from Dr J. S. Wiberg. The T4 mutants amN134 (gene 33) and amBL292 (gene 55), were provided by Dr A. Belle. The double mutant amC42 x amBL292 was constructed and identified by the complement&ion spot test technique t Abbreviations
and triphosphate,
used:
FU,
respectively.
5-fluoroaracil;
FUMP,
FUDP,
FUTP,
5.fluorouridine
mono-,
di-
REGULATION described by Wiberg dz Buchanan, 1964).
(1966). Phages
OF T4 RNA were grown
341
SYNTHESIS
and harvested
as described
earlier
(Skolci
(b) ChernicaLs [MethyZ-3H] thymidine (2.0 to 5.0 c/m-mole) and [6-3H]5-fluorouracil ~870 me/m-mole) was obtained from The Radiochemical Centre, Amersham, England. ~-[~~C]Leucine (uniformly labelled, 250 me/m-mole) was purchased from New England Nuclear Chemicals, Dreieichenhain, Germany and 2-14C-labelled 5-fluorouracil (22 me/m-mole) from Calbiothem, Luzern, Switzerland. Unlabelled 5fluourouracil was a gift from F. Hoffman-La Roche & Co., Basle, Switzerland. (c) Media All experiments were carried out in a low phosphate mineral salts medium of the following composition/l.: 0.5 g NH,Cl, 0.5 g (NH4)zS04. 5.0 g NaCl, 0.5 g KCl, 0.203 g MgCl,.6H,O, 14.7 mg CaCl,.2H,O, 0.178 g Na2HP04.2H,0 and 6.25 g Tris, pH was adjusted to 8.0 with M-HCl and after amoclaving the medium was supplemented with glucose 5 g/l. and FeCI, to 10m5 r+r, Bottom agar for phage plating, contained/l.: 10 g Agar (Bacto), 13 g Bactotryptone, 8 g NaCl, 2.3 g sodium citrate.2H,O, 1.3 g glucose. Top agar for phage plating contained/l.: 6 g Agar (Bacto), 10 g Bactotryptone, 8 g NaCl, 2.3 g sodium citrate.2Ha0, 3 g glucose, 20 mg L-tryptophan. Dilution fluid for phage contained/l. : 10 g Bactotryptone, 5 g Nacl, 20 mg L-tryptophan. (d) Infected
cells
Bacteria were grown to 5 x lO*/ml. at 37°C in the mineral salts medium, (supplemented with uridine 10 pg/ml. for OK302, generation time about 53 mm). The harvested cells were washed once and resuspended to about 1.5 x 1 Og/ml. in the same medium supplement& with L-tryptophan 10 pg/ml., thymidine 20 pg/ml. and FU 10 pg/ml. or uridine 10 pg/ml. Infection was performed by the addition of 7 to 9 phages per bacterium. After 3 min of adsorption the infected cells were diluted (usually lo-fold) into warmed, aerated mineral salts medium containing radioisotope and FU or uridine. All experiments with FU were performed in the presence of thymidine to neutralize the very strong inhibitory effect on thymidylate synthetase by 5-fluorodeoxyuridine monophosphate, which could form intracellularly (Cohen, Flaks, Barrier, Loeb & Lichtenstein, 1958). Phage adsorption, bacterial survival, number of infective centres and burst size were determined by standard techniques. Adsorption was usually better than 99% at the end of the 3-min adsorption period and bacterial survival less than 1 ?A. In all experiments phage infection is coumed as time zero. (e) Measurement
of RNA,
DLNA and protein
synthesis
The synthesis of DNA and protein was determined by the cumulative incorporation of [3H:]thymidine and L-[%]leucine, respectively, into acid-insoluble product. Similarly the incorporation of FU into RNA was studied by the cumulative incorporation of 14C- or 3H-labeled 5-fluorouracil into acid-insoluble product. In the latter case there would presumably be no interference from FU being incorporated into phage DNA, since that would require the removal of the &jZuorine, and 5-fluorouracil is known to be quite stable intracellularly (Horowitz & Chargaff, 1959). Samples of 1.0 or 1.5 ml. were withdrawn at different times from the aerated, phage-infected cultures and precipitated in 5.0 ml. and 7.5 ml., respectively. of 6%, cold trichloroacetic acid. The precipitates were collected on membrane or glass fibre filters (Gelman, Metricel GA-6 and Whatman GF/C, respectively). Radioactivity was measured in a Packard 3375 scintillation counter after immersing the dried filter in naphthalene-dioxane scintillation fluid (Bray, 1960). When 14C and 3H were measured in the same sample, spectrometer settings were used which gave less than 0.5% of the tritium activity in the i4C channel. Since the aH/14C ratio of the incorporated activities usually varied between 05 to 8.0, this tritium overlap was ignored. About 3076 of the i4C counts appeared in the 3H channel and appropriate corrections were made.
342
0. (f) Measurement
SKC)LD
of cellular
ti-fhorouracd
nucleotido
pools
The incorporation of 3H-labelled-5-fluorouracil into the nucleoside mono-, di- and triphosphate pools of phage-infected cells was determined after nucleotide separation by high voltage electrophoresis of perchloric acid extracts. At different times after infection, 2-m]. samples were withdrawn from the infected cultures containing 3H-labelled FU, and filtered on a membrane filter (Gelman, Metricel GA-6). The cells were rapidly washed on the filter wit,h 2 x 1 ml. of mmeral salts medium without glucose, and finally suspended in 1 ml. of cold 0.4 M-perchloric acid. After centrifugation the supernatant fraction was neutralized with 1 M-KOH and the perchlorate precipitate centrifuged off. Samples of this supernatant fraction were subjected to electrophoresis on Whatman 3 MM paper in a 0.05 M-citrate buffer, pH 4.6, for 2 hr at 60 v/cm. Samples of UMP, UDP and UTP could be used as references, since initial test runs showed these nucleotides to move identically with the corresponding FU-nucleotides at the pH used. The nucleotide spots were localized by ultraviolet light, cut out, the paper discs immersed in 20 ml. of naphthalene-dioxanc scintillation fluid (Bray, 1960), and counted in a Packard scintillation counter.
3. Results (a) Inhibition
of bacteriophuge production by 5-jluorouracil at high temperature
The effect of FU on T4 phage production in E. coli B at different temperatures is demonstrated in Table 1. In the presence of 10 pg FU /ml. and at an elevated temperature (42”C), phage production is seen to be completely abolished. The same concentration of FU does, however, allow phage production at lower temperatures. The burst size is thus decreased by only 30% at 26°C. The heat-sensitizing effect of FU on phage development is reversible, which is shown in Figure 1. It can be seen that phage production in the uracil-requiring mut,ant OK302 in the presence of FU recovers if the temperature is shifted from high (41°C) to low (3O”C), and with increasing efficiency the earlier the shift is made. The two lower curves of Figure 1 are controls, without temperature shift and run at 41 and 43”C, respectively. Some production of phage can be observed late in the infection period at 41”C, whereas the inhibition is complete at 43°C. That the high temperature per se did not affect phage development in OK302 is shown in Figure 2, which demonstrates intracellular phage production at 41G”C TABLE
1
Temperature sensitivity of bacteriophage production in the presence of 5-jluorouracil Temperature
42 35 30 26
(“C)
Burst
(% of control)
0 20 35 70
Cells, E. wli B, were grown and infected with T4 as described. The adsorption mixture containing 10 pg FU/ml.; 20 pg thymidine/ml. and cells (1.2 x 10’O/ml.) was warmed to the experimental temperature before addition of phage. After phage adsorption for 3 min without aeration, the infected complexes were diluted lo-fold into aerated mineral salts medium of the experimental temperature and containing 10 pg FU/ml. and 20 pg thymidine/ml. Lysis was induced with chloroform after 60 min of incubation. The results are given as percentage of control burst size, obtained in t,he absence of FU at all temperatures (130 to 140 per infective centre).
REGULATION
OF T4 RNA
343
SYNTHESIS
4041° c +30°
c
30-
//Ii
a,&(,&
IO
20
30
40
50
60
70
80
Time (mln)
FIG. 1. Reversibility of FU effect by temperature shift. Cells, OK302, (uracil-) were grown and infected as described. The adsorption mixture which contained 10 pg FU/ml. and 20 pg thymidinelml. was warmed to 41°C before phage infection. The infected cells were diluted lo-fold into mineral salts medium at 41°C containing 10 pg FU/ml. and 20 pg thymidine/ml. Shifts to 30°C were performed at the indicated times. At the different points samples were withdrawn and lysed with chloroform. Results are given as number of phage produced per bacterium. The two lower curves are from experiments without temperature shift. --x--x--,-A--A--, and -O--O--, 41°C; --n--n--, 43’C.
(lysis induced with chloroform). The formation of mature phage was observed to start 10 to 12 minutes after infection and proceeded almost linearly until 40 minutes after infection. (b) Rescue by uridine of bacteriophage production inhibited by 5-jluorouracil at a high temperature Not only shift in temperature but also the addition of uricline was shown to reverse the inhibition of phage production by FU at a high temperature. This is most simply explained as a chasing out of FU by uridine from the metabolically labile phage mRNA. The phenomenon is demonstrated in Figure 3, where the lowest curve shows an almost complete inhibition of phage production at 41.5% in the presence of FU 10 pg/ml. When uridine was added to a final concentration of 150 pg/ml. at seven minutes after infection, phage production was observed to approach the values of the control without FU. Addition of uridine at later times after infection gave a gradually diminishing response. It thus seemed as if FU was chased from phage RNA wit)h decreasing efficiency at later times after infection.
I 0
IO
20
30
40 50 Time (mid
60
70
8C
FIG. 2. Phage production at high temperature in the uracil-requiring E. co& B strain OK302. Phage infection was performed as described. The temperature W&S 41.5”C, and the adsorption mixture containing 10 pg uridine/ml. was warmed before the addition of phage. After adsorption the infected cells were diluted IO-fold into mineral salts medium at 41.5”C containing 10 pg uridine/ml. At the indicated times, samples were withdrawn, lysed with chloroform and assayed for phage. The results are given as number of phages produced per ml. of aerated growth culture.
160-
60-
0
1
60
80
100
Time (min)
FIG. 3. Uridine rescue of phage production inhibited by FU at a high temperature. The experiments were performed as described in the legend to Fig. 1 with strain OK302 as host, and at 41.5’C. The upper curve represents a control without FU but with 10 pg uridine/ml. At the indicated times, uridine was added to a final concentration of 150 pg/ml. Results are given as phages produced per input bacterium.
REGULATION
(c) Inhibition
of bacteriophage
OF T4 RNA
DNA
345
SYNTHESIS
synthesis by 5-jluorouracil
at high temperature
The inhibiting effect of FU on phage production at a high temperature could br explained by its interfering witch early protein formation via its incorporation ink, early mRNA. This interpretation is supported by the experiment shown in Figure 4. Phage-specific DNA synthesis, measured as the cumulative incorporation of radioactive thymidine into acid-insoluble product, was seen to be almost completely inhibited by the presence of FU at 41°C. The inhibition could, however, be reversed b> shifting the temperature from 41 to 30°C at seven and nine minutes, respectively, after infection. The rescue of DNA synthesis was significantly more efficient with temperature shift at seven minutes than at nine minut.es. The FU-inhibition of phage DNA synthesis at a high temperature is not a conscquencr of a general toxic effect of FU on the cells. This is demonstrated in Figure 5(a), I
I
I
I
I
8000 -
2000 -
Time (min) Fm. 4. Inhibition
of phage DIiA
synthesis by FU at high temperature and its reversion by temperature shift. Cells (OK302) were grown and infected as described. Synthesis of DNA was measured by the cumulative incorporation of [sH]thymidine into acid-insoluble product as described. The phage was allowed to adsorb for 3 min at 41.5% and the infected cells were then diluted lo-fold into mineral salts medium at 41.5”C containing [eH]thymidine 20 pg/ml; 1 PC/ml. and FU 10 pg/ml or uridine 10 pg/ml. (control). The values along the ordinate are expressed as counts per min. incorporated per 1.5ml. sample. Phage infection at time zero. -O-O-, +- FU, no temperature shift; -O-O-, + FU, temperature shift to 30°C at 9 min; -A-A--, + FU, temperature shift to 30°C at 7 min; -xx--, control with uridine, temperature shift to 30°C at 13 min.
3ir
I IO
I 20
30
40
I 50
60
Fm. 5. Absence of toxic effects by FU on host cells. (a) Cells (strain OK302) were grown, harvested, washed and resuspended as described. The experiment was started by diluting the uninfected cells lo-fold into mineral salts medium warmed to 42’C containing 20 pg [3H]thymidine/ml., 1 pc/ml., and 10 pg FU/ml. or 10 pg uridine/ml. (control). Values along the ordinate are expressed as counts per min per l&ml. sample. - x - X -, -+ 10 pg FU/ml.; -O-O-, control with 10 pg uridine/ml. (b) Cells (OK302) were prepared as in (a). The experiment was started by diluting the uninfected cells IO-fold into mineral salts medium containing 20 pg thymidine/ml. and 10 pg FU/ml. or 10 pg uridine/ml. (control). After incubation at 41°C for 20 min the cells were cooled, concentrated lo-fold by centrifugation and infected by phage. After adsorption for 3 min at 41°C the infected cells were diluted lo-fold into mineral salts medium at 41°C containing 20 pg [3H]thymidine/ml., 1 PC/ml. and 10 pg uridine/ml. but no FU. Values along the ordinate are expressed as counts per min per 1.5ml. sample. Phage infection at time zero. - x - X-, cells preincubated + 10 rg FU/ml.; -O-O-, control cells preincubated with 10 pg uridine/ml.
where the DNA synthesis in uninfected cells of the uracil-requiring E. coli B strain OK302 is seen to be completely unimpaired by the presence of FU at 42°C. That FU really entered the RNA under these conditions is shown below (Fig. lO( b)). The decrease in C3H]thymidine incorporation observed at 30 to 40 minutes after the addition of labelled precursor is probably explained by the enzymic degradation of thymidine to thymine, which is a very poor precursor for DNA synthesis in uninfected but not in T-even phage infected cells (Kemmen & Strand, 1967; Munch-Petersen & Vilstrup, 1969, Abstracts, Fed. EUTO~.Biochem. Sot., no. 873, &lad&d). Another experiment to check for the possible toxic effect of FU on the host cells is shown in Figure 5(b). In this c&se the uninfected cells were pretreated with FU (10 pg/ml.) at 41°C for 20 minutes, centrifuged, washed and finally resuspended in the absence of FU and infected by phage at 41°C. The FU-pretreated cells were observed to support phage DNA synthesis just as well as the control.
REGULATION
OF
T4 RNA
347
SYNTHESIS
The effect of FU on protein and DNA synthesis, respectively, was furthermore directly compared in phage-infected cells. In the experiment presented in Figure 6, phage DNA and phage protein synthesis were measured simultaneously in cells infected at 41.5”C and in the presence of FU. Rate and extent of protein synthesis is seen I
150 -
I
I
I
3H activity
A-A-A
A-A-A-
IO
(a)
Time (mm)
30
20
40
(b)
Fro. 6. Effect of FU on protein synthesis at a high temperature. The experiment was performed at 41.5”C as described in the legend to Fig. 4. The infected cells (OK302) were diluted lo-fold into mineral salts medium containing 20 pg [3H]thymidine/ml., 1 &ml.; 10 pg [W]leucine/ml., 0.05 PC/ml. and 10 pg FU/ml. or 10 pg uridine/ml. (control). In one control experiment the dilution medium also contained 20 pg chloramphenicol/ml. The 3H- and 14C-radioaotivities were separated in the scintillation counter as desoribed in the Materials and Methods section. The values along the ordinate are expressed as counts per min incorporated per 1 ml-sample of sH and W activity, respectively. Phage infection at time zero. control with 10 pg uridine/ml.; -s--O-, + 10 pg FU/ml.; -A-A-, control -x-x-, with 10 pg uridine/ml. and 20 pg chloramphenicol/ml.
to be indistinguishable from those of the control with uridine instead of FU. Phage DNA synthesis was markedly inhibited by FU, although not completely, in this experiment (of. curve obtained in the presence of chloramphenicol). The quantity of proteins produced in the presence of FU was thus unimpaired. Their quality on the other hand seemed to be changed as reflected by the inability of the early enzymes produced, to support a normal phage DNA synthesis. (d) Rescue by uridine of bacteriophage DNA synthesis inhibited by 5-$uorouracil at a high temperature The pronounced inhibitory effect of FU on phage DNA synthesis at a high temperature could be reversed by the addition of uridine. As demonstrated in Figure 7 this reversion became, however, less pronounced, the later uridine was added after infection. Thus uridine addition at seven minutes gave a DNA synthesis which was about
348
0.
SKC)bD
Fra. 7. Uridine rescue of phage DNA synt,hesis inhibited by FLJ at a high kmperature. The experiment was performed as described in the legend to Pig. 4 and at 41’C. The infecked C& (strain 0X309) were diluted lo-fold into mineral salts medium containing 20 pg [W]thymidine/ml., 1 hq’rnr. and 10 pg l?U/ml. OP 10 ~6 uridinejml. (control). At the times indicated on each curve, uridina was added to the infected cultures to a final concentration of 150 pg,knl. The values along the ordinate are expressed as counts per min incorporated per 1.5.ml. ssmpls. Pbage infection at time zero. -o-O--, + FU, 160 pg uridine/ml. added at - x - x -, Control wit)h lO,ug uridine/ml.; 7min; -A--&--, + FU, 150 @ uridinc + FU, 150 ~IF;nridine/ml. added at 9 min; --n--A--, {ml. added at 18 min; --+-a--, + EZT, 150 pg uridine/ml. added at 24 min.
that of the control. Uridinc addition at nine minutes and 22 minutes gave a synthesis of 14 and ‘I%, respectively. The reversion of IMA synthesis inhibition thus foIlowed a pattern similar to that for uridine rescue of EJ-inhibited phage production shown jn Figure 3. The discrepancy between appa,rent efficiency of uridine rescue of phage production (Fig. 3) and of DNA synthesis (Fig, 7) is probably explained by tke fact that cumulative incorporation of ~3H]thymidine measured total DNA synthesis, e.g. alao Ghe formation of DNA which will never be incorporated into pbage. The relatively large phage production even after relatively long periods of FU inhibition (Fig. 3) would then represent an eficient utilization of the small amounts of DNA formed during FU inhibition (cf. Fig. 6). The measurement of total DNA synthesis
SO$/, of
REGULATION
OF T4 RNA
SYNTHESIS
340
would then, according to this argument, more closely reflect the total capacity of the early enzymes formed. The rapidly diminishing efficiency of uridine rescue of FUinhibited DNA synthesis is interpreted to reflect a decreasing synthesis of early mRNA. Uridine could chase out FU from early mRNA only as long as the latter is actively turning over. A diminishing early mRNA synthesis would support the synthesis of decreasing amounts of early enzymes, and successively of DNA. (e) Early mRNA synthesis in gene 33 and gene 55 TQ-natiants, resected us uridine rescue of 5-$uorouracil-inhibited DNA fornaation The amber mutants amN134 (gene 33) and amBL292 (gene 55) are known as maturation deficient, i.e. they will perform nothing or very little of the late functions, although they are capable of DNA synthesis (Bolle et al., 19686). Since these mutants seem to have a defect in the normal change from early to late events during phage development,, the kinetics of their early mRNA syntjhesis was studied wit*h the method described in the previous section. They were thus tested for uridine reversion of the
Time
FIG. The control uridine
(min)
8. Uridine rescue of mutant amBL292 DNA synthesis inhibited by FU at a high temperature. experimental procedure was identical to that described in the legend to Pig. 7. - x - x --, with 10 pg uridine/ml.; -A-A--, -I- FU, uridine added at 14.5 min; -A-A-, + FIT, added at 22 min; -O--c--, + FU, uridine added at 25 min.
0.
350
SKC)LD
FU inhibition of their DNA synthesis. With the mutant amBL292 the decrease in reversion efficiency with time of uridine addition was much less pronounced t’han with wild type as shown in Figure 8 (cf. Fig. 7). Addition of uridine at 22 to 25 minutes after infection in the presence of FU at 41°C could thus rescue substantial amounts of mutant DNA synthesis. A very similar behaviour was found for the mutant anziV134 (gene 33) (data not shown). Figure 9 shows a comparison of the uridine rescue of
I 0
I IO
I
J
20 Time (min)
FIQ. 9. Comparison of uridine rescue of FU-inhibited DNA synthesis in anaBL292 and T4 infection. Replot of data from Figs 7 and 8. Incorporation of [aH]thymidine between 40 and 60 min after mutant and wild-type phage infection, respectively, in FU inhibited cultures. Values are expressed as percentages of the corresponding values for controls without FU and plotted Z)WSW time of uridine addition. ---AA-, amBL292 infection; - x - x -, T4 + infection.
BU-inhibited DNA synthesis after amBL292 (gene 55) and wild type T4 infection, respectively. The cumulative 13H]thymidine incorporation between 40 and 60 minutes. after infection in B-m-inhibited cultures is plotted versus time of uridine addition. The late time period for DNA determination was chosen to avoid interference with the late addition times of uridine. A clear difference between mutant and wild type infection can be seen. When uridine was added at, for example, 14.5 minutes after infection 96% of mutant DNA synthesis was rescued in contrast to only 5 to 8% of that of the wildtype. (f) Incorporation
and chase of 5-$uorouracil in bacterio@uzge-infected non-infected
and
cells
A direct demonstration of the chasing out of FU from phage mRNA with uridine is shown in Figure IO(a). The radioactivity incorporated from l*C-labelled FU into acidinsoluble RNA is seen to disappear rapidly after the addition of uridine. The chase is not complete, however, which is interpreted as a reutilization of labelled FU nucleotides for the synthesis of late phage mRNA. A chase performed at 15 minutes gave a similar pattern (data not shown). In one experiment with uridine chase at seven minutes, phage DNA synthesis was measured simultaneously. It can be seen, that the initially inhibited DNA synthesis was released about 11 minutes after uridine addition which corresponds to thr time period Mn-ten infection and start’ of DNA synthesis in
REGULATION
0
IO
’
’
20
30
OF T4 RNA
40
0
351
SYNTHESIS
IO
20
30
40
Time (mln)
(a) (b) 10. Chasing of FU from RNA of phage-infected and uninfected cells (OK302). (a) The experiment was performed as described in the legend to Fig. 4 and at 41°C. The infected cells (strain OK302) were diluted lo-fold into mineral salts medium containing 20 pg thymidine/ml. FU/ml., 0.05 pc/ml. At the (3H-labelled, 1 &ml., in one experiment) and 10 pg l*C-labelled indicated times uridine was added to the infected cultures to a final concentration of 150 pg/ml. 14C- and 3H-activities were separated in the scintillation counter. The values along the ordinates are expressed as counts per min incorporated per l-ml. sample. Phage infection at time zero; - x - x -, no uridine chase; -O-O-, uridine added at 7 min; -n-n-, uridine added at 12 min; --A--A--, DNA synthesis ([3H]thymidine incorporation) in the experiment with uridine addition at 7 min. (b) Bacterial cells (OK302) were prepared as described. The experiment was started by diluting the cells IO-fold into mineral salts medium at 30°C containing 20 pg thymidine/ml. and 10 pg 3H-labelled FU/ml., 1 PC/ml. The values along the ordinate are expressed as counts per min incorporated per l-ml. sample. - X-X -, no uridine chase; -O-O--, uridine added to 150 pg/ml. at 8.5 min. FIG.
normal infection (cf. Fig. 7). Phage-infected cells incorporate little l*C-labelled FU int.0 acid-insoluble product later than ten minutes after infection (Fig. 10(a), upper curve). In contrast, uninfected cells (Fig. 10(b)), which synthesize stable RNA, continue to incorporate large amounts of 3H-labelled FU for the studied time period of 40 minutes. In concurrence with this observation the radioactivity incorporated from l*C-labelled FU into the RNA of uninfected cells could not be chased with uridine (Fig. 10(b)).
(g) Rate of early mRNA synthesis at different times after bacteriophage infection In order to obtain a more direct evaluation of the changing intensity in early mRNA formation with time after infection, the rates of early mRNA synthesis were mea.sured by determining the short-time (1 min) incorporation of l*C-labelled FU into acidinsoluble product after infection with phage and’42 (gene 1). This mutant is deficient in dHMP-kinase and cannot induce any formation of DNA in E. coli B. Since there is 24
352
0.
SKi)LD
Time (mid FIQ. 11. Rates of RNA synthesis at different times after infection with mutants and wild type of T4. Cells of E. coli B were grown and infected as described, but FU was not added to the adsorption mixture. After adsorption for 3 min at 37°C the infected cells were diluted lo-fold into mineral salts medium at 37°C containing 20 pg thymidine/ml. At the different times sH-labelled FU was added to 10 pg/ml. and 1 &nl. Samples of 1 ml. were withdrawn and precipitated with trichloroacetic acid after 1 min. The values are plotted as percentages of the 1-min incorporation values for eH-labelled FU addition at 3 min. These values were in counts per min per 1 ml.-sample: 207 for amC42, 135 for anaC42 x arnBL292 and 126 for T4+.
no DNA synthesis in amC42-infected E. coli B, there is no formation of late mRNA (Bolle et al., 196%). This means that any incorporation of l*C-labelled FU into acidinsoluble product after infection must represent early mRNA. In Figure 11 some experiments are demonstrated, in which the incorporation of 14C-labelled FU added at different time points after infection is measured during one minute periods. The data are plotted as percentages of the incorporation obtained at three minutes, when the incorporation was maximal. A comparison of the diagrams for amC42 (top part) and T4+ (bottom part) in Figure 11 shows that the short-time incorporat,ion of l*C-labelled FU diminishes rapidly with time of pulse addition in cells infected with the mutant. Pulses given at 15 and 20 minutes, respectively, hardly give any observable incorporation with the mutant, which indicates a rapidly diminishing synthesis of early mRNA. The incorporation with T4 at 20 minutes is about 35% of that at three minutes, representing mainly late mRNA synthesis. In Figure 11 is also shown an
REGULATION
OF T4 RNA
353
SYNTHESIS
experiment with a double mutant amC42 x amBL292 (gene I x gene 55). It can be seen that the presence of both mutations in the same genome prolonged the time period of early mRNA synthesis in the mutant-infected cells. The pulse incorporations at 15 and 20 minutes were 58 and 2Oo/oof that at three minutes, respectively, and thus substantially higher than the corresponding values for the gene 1 single mutant. These results imply that gene 55 is in some way connected to the regulation of early mRNA synthesis. The low pulse incorporation of 3H-labelled FU into phage RNA at later times with the amC42 mutant could be due to a diminishing uptake of the labelled base into the nucleotide pool. To rule out this possibility, labelled 5-FU was added to amC42-infected cells at 3 and 12 minutes, respectively, and its incorporation into nucleoside mono-, di-and triphosphate pools was determined by analysis of extracts with high voltage electrophoresis. The results are presented in Table 2. It can be seen that the general TABLE
2
Uptake of 3H-labelled PU into the nucleotide pools of amC42- and T4+-infected cells 3H-labelled Time (min) 6 11 23
FUMP 100 245 1112
3H-labelled
FU added at 3 min amC42 FUDP 238 473 525
FUTP 415 1012 1780
T4 FUTP 560 1462 1670
Tim0 (mm) 18 23 32
FUMP 275 415 320
FU added at 12 min amC42 FUDP 388 440 507
FUTP 762 810 1330
T4 FUTP 830 1430 2090
E. coli B cells were grown and infected with phage as described, but FU was not added to the adsorption mixture. After phage adsorption for 3 min at 37°C the infected cells were diluted 6.4fold into mineral salts medium at 37% containing 20 pg thymidine/ml. and 9.5 ng FU/ml. At 3 min and 12 min, respectively, after infection, 3H-labeled FU was added to 7.8 pc/ml. At the indicated times after infection 2-ml. samples were withdrawn and analysed as described in the Materials and Methods section under (f). The values are expressed as counts per min per 3.5 x lo7 cells.
incorporation pattern of 3H-labelled FU into the different nucleotide pools is not changed dramatically when the labelled precursor is added at 12 minutes after infection as compared to addition at three minutes. Formation of FUTP, which is the immediate precursor of FU-containing RNA, is seen to continue during the period 12 to 32 minutes after infection. The uptake of FU into the nucleotide pools of amC42infected cells is thus not restricted at late times after infection. For comparison the 3H-labelled FU uptake into the FUTP pools of T4+-infected cells is also shown in Table 2.
4. Discussion In these experiments I have investigated the regulation of early mRNA formation in T4 phage-infected E. coli B cells. Two different approaches were used. One was functionally to “label” newly formed mRNA specifically involved in phage-specific DNL4 synthesis. The other was to measure rates of early mRNA synthesis by radioactive pulse labelling. Gros & Naono (1961) showed that E. coli cells grown in the presence of FU produced an alkaline phosphatase which was much less thermostable than the enzyme formed in
354
0.
SKOLD
the absence of the analogue. They concluded that FU was incorporated into the mRNA corresponding to alkaline phosphatase. A similar effect was observed in the present experiments, where the presence of FU at a high temperature was seen to abolish phage production and more specifically phage DNA synthesis. The known inhibitory effect of the FU-deoxynucleotide on thymidylate synthetase (Cohen et al., 1958) was neutralized by the addition of thymidine in all experiments. The observation that both DNA formation and phage production could be restored by shifting to a lower temperature, indicates a FU-induced heat-lability in one or several of the enzymes responsible for phage DNA synthesis. This interpretation is further supported by the finding that bulk synthesis of phage proteins is unimpaired under conditions when FU severely restricts phage DNA formation. That the effect of FU is directed towards enzymes being synthesized is furthermore indicated by the insensitivity of host bacterial DNA synthesis to the presence of FU at a high temperature. Both phage-infected and uninfected bacteria readily took up FU into RNA. I conclude that the inhibitory effect of FU on phage DNA synthesis is a consequence of its incorporation into phage mRNA, where it introduces coding errors, which are finally expressed as a decreased thermostability of early phage enzymes. Inhibition by FU of both phage production and phage DNA synthesis at high temperature was reversed by the addition of uridine, which efficiently enters labile RNA as uracil. This is interpreted as a metabolic dilution of FU from mRNA to allow the formation of mRNA without coding errors and consequently of normal early enzymes. The effect on phage DNA synthesis of this metabolic chase of FU from mRNA could however, be observed only as long as active synthesis of that mRNA fraction which is responsible for the formation of DNA-synthesizing enzymes proceeds. The experiments demonstrated a decrease in the efficiency of uridine rescue of both phage production and phage DNA synthesis with time after infection. This indicates a gradual decrease in the synthesis of mRNA coding for early enzymes, because the later uridine is added, the less early enzymes in heat-stable form would then eventually become available for DNA synthesis. The pattern of uridine rescue of DNA synthesis was quite different after infection with mutants of phage T4 carrying lesions in either one of genes 33 and 55. In these cases, DNA synthesis at high temperature could be rescued from FU-inhibition at much later times than in wild-type infection, indicating a much slower decrease in early mRNA synthesis. This is in accordance with the results of Hosoda & Levinthal (1968), who could show a delay in the time when early protein synthesis is normally stopped after infection with genes 33 and 55 mutants, respectively. Since the uridine rescue is mediated by the early mRNA synthesis, I conclude that the experiments indicate the presence of a transcriptional regulation mechanism which terminates the synthesis of early, DNA-synthesizing enzymes, and that this mechanism is dependent on an undisturbed function of genes 33 and 55, respectively. The interpretation of these experiments relies on the efficiency of the uridine chase of FU from mRNA at different times after infection. For this reason the incorporation kinetics of FU and of uridine into phage mRNA was given special attention. FU into phage mRNA at the high The cumulative incorporation of l*C-labelled temperature was essentially finished at about ten minutes after infection, at which time a plateau was reached (Fig. 10(a)). Since very little (Weiss, Hsu, Foft & Soherberg, 1968) stable RNA is formed after phage infection, this plateau should represent
REGULATION
OF T4 RNA
SYNTHESIS
355
an equilibrium between uptake of 14C-labelled FU into mRNA and mRNA degradation. According to Nierlich (1967) the uptake of a radioactive RNA precursor would take place only as long as net synthesis of RNA proceeds. The initial branch of the 14Clabelled FU incorporation curve (Fig. 10(a)) should then represent an expansion of the pool of phage mRNA. That an increase in the cellular contents of labile mRNA does occur in the early period after infection is supported by the findings of Landy & Spiegelman (1968) and Kleppe & Nygaard (1969). The plateau of the incorporation curve has a slight slope of increase, which, however, extends far beyond the time (10 to 12 min) when early mRNA formation according to the uridine rescue experiments would be essentially finished. The RNA synthesis corresponding to the latter portions of this plateau would then consist of late mRNA synthesis. It is, however, known that no late mRNA synthesis could be initiated without preceding phage DNA synthesis (Bolle et al., 19683), which in this case ought to be inhibited by FU at the high temperature. The synthesis of late mRNA is explained here as a result of an incomplete FU-inhibition of phage DNA formation (could be seen in Fig. 6). It is known that a small amount of DNA synthesis, for a short period during phage development, is sufficient to allow late mRNA to be formed (Bolle et al., 19686). This interpretation is supported by the observation that FU-label could be chased from phage RNA as late as 12 to 15 minutes after infection. At these late times, the uptake of RN-4 precursors would then be effected through the slow expansion of the late mRNA pool and possibly also by expansion of the nucleotide pools (discussed below). I conclude that FU can be efficiently chased from phage RNA by uridine during the initial 15 minutes of the infection period, but that only chases earlier than 9 to 12 minutes can rescue the synthesis of early enzymes involved in phage DNA synthesis. The determinations of total phage RNA synthesis, by short-time labelling at different times after infection, also indicated the presence of a transcriptional regulation mechanism which switches off early mRNA synthesis. The rates of total RNA formation thus decreased rapidly with time after infection with mutant an&‘42 (gene l), which can induce only early mRNA synthesis. The decrease in early mRNA synthesis was, however, much slower in a double mutant containing a mutation also in gene 55. These results support the conclusion that gene 55 is involved in the transcriptional regulation of early mRNA formation. The validity of the short-time labelling experiments depends on the ability of the labelled precursor to enter the nucleotide pools. According to Nierlich (196’7), labelled precursor would be taken up into the nucleotide pools only as long as there is a net utilization of nucleotides for RNA synthesis under conditions where nucleotide pools do not expand. In the present experiments where presumably no net synthesis of RNA occurs this would mean that 14C-labelled FU would enter the nucleotide pools only as long as the pool of labile phage mRNA expands. If this expansion ended early (cf. discussion above of experiment in Fig. 10(a)) the interpretation given above would be invalidated, It was, however, shown by the direct analysis of nucleotide labelling, that the uptake of label into nucleotides continued at, a substantial rate as late as 23 to 32 minutes after infection, under conditions when only early mRNA formation takes place. This period is far beyond the time when early mRNA synthesis is shut off as discussed above. These results could be explained by an expansion of the nucleotide pools, which is known to occur in bacteria after the arrest of net RNA synthesis (Edlin & Stent, 1969).
356
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This research was supported by a grant from the Swedish Medical Research Council. REFERENCES Belle, A., Epstein, R. H., Salser, W. & Geiduschek, Bolle, A., Epstein, R. H., Salser, W. & Geiduschek, Bray, G. A. (1960). Analyt. Biochem. 1, 279. Cohen, S. S. (1948). J. BioZ. Chem. 174, 281. Cohen, S. S. (1963). Ann. Rev. Biochem. 32, 83. Cohen, S. S. (1968). Virus-induced Enzymes, New Cohen, S. S., Flaks, J. G., Barner, H. D., Loeb, M. Acad.
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E. P. (1968a). J. Mol. Biol. 31, 325. E. P. (1968b). J. Mol. Biol. 33, 339.
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Edgar, R. S. & Wood, W. B. (1966). Proc. Nut. Au&. Sci., Wash. 55, 498. Edlin, G. & Stent, G. S. (1969). Proc. Nut. Acad. Sci., Wash. 62, 475. Gros, F. & Naono, S. (1961). In Protein Biosynthesis, ed. by R. J. C. Harris, p. 195, New York : Academic Press. Hall, B. D., Nygaard, A. P. & Green, M. H. (1964). J. Mol. BioZ. 9, 143. Horowitz, J. & Chargaff, E. (1959). Nature, 184, 1213. Hosoda, J. 8: Levinthal, C. (1968). Viirology, 34, 709. Kammen, H. 0. & Strand, M. (1967). J. BioZ. Chem. 242, 1854. KarlstrGm, 0. (1968). J. Bact. 95, 1069. Khesin, R. B., Gorlenko, Zh.M., Shemyakin, M. F., Bass, I. A. & Prozorov, A. A. (1963). Biokhimya, 28, 1070. King, J. & Wood, W. 13. (1969). J. Mol. BioZ. 39, 583. Kleppe, R. K. & Nygaard, A. P. (1969). Biochim. biophys. Actu, 190, 202. Kornberg, A., Zimmerman, S. B., Kornberg, S. R. & Josse, J. (1959). PTOC. Nat. Acad. Sci.,
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Landy, A. & Spiegelman, S. (1968). Biochemistry, 7, 585. Nierlich, D. P. (1967). Science, 158, 1186. Nomura, M., Okamoto, K. & Asano, K. (1962). J. Mol. BioZ. 4, 376. Nomura, M., Witten, C., Mantei, N. & Echols, H. (1966). J. Mol. BioZ. 17, 273. Salser, W., Belle, A. & Epstein, R. (1970). J. Mol. BioZ. 49, 271. SkGld, 0. & Buchanan, J. M. (1964). Proc. Nat. Acad. Sci., Wash. 51, 553. Weiss, S. B., Hsu, W., Foft, J. W. & Scherberg, N. H. (1968). Proc. Nat. Acad. Sci., Wash. 61, 114.
Wiberg, J. S. (1966). Proc. Nut. Acad. Sci., Wash. 55, 614. Wiberg, J. S., Dirksen, M., Epstein, R. H., Luria, S. E. & Buchanan, Nat. Acad. Sci., Wash. 48, 293.
J. M. (1962). Proc.