To study the mechanism by which bacteriophage T4 inhibits the synthesis of ... The kinetics of cessation of fl-galactosidase synthesis after T4 infection in-.
Vol. 17, No. 2 Printed in U.S.A.
JOURNAL OF VIROLOGY, Feb. 1976, p. 326-334 Copyright @ 1976 American Society for Microbiology
Bacteriophage T4-Induced Shut-Off of Host-Specific Translation STEFAN B. SVENSON* AND OLLE H. KARLSTROMI Department of Microbiology, Faculty of Pharmacy, University of Uppsala, S-751 23 Uppsala, Sweden Received for publication 18 June 1975
To study the mechanism by which bacteriophage T4 inhibits the synthesis of inducible host enzymes we measured the formation of f-galactosidase from preformed lac mRNA. fl-Galactosidase was induced with isopropyl-f-D-thiogalactopyranoside in the presence of 7-azatryptophan, a tryptophan analogue that is incorporated into proteins and renders the fl-galactosidase formed inactive. The accumulated lac mRNA was measured by capacity to form active fl-galactosidase after a chase of the analogue with excess tryptophan. After T4 infection the ability to form f-galactosidase from the preformed lac mRNA was rapidly lost even when T4 infection took place in the presence of rifampin. This restriction was dependent on the multiplicity of infection. At a multiplicity of infection of 8.6, 90% of the ability to express preformed lac mRNA was lost within 30 s. The kinetics of cessation of fl-galactosidase synthesis after T4 infection indicate that infection blocks initiation of lac mRNA translation. T-even bacteriophages infecting Escherichia coli cause an inhibition of host-specific macromolecular synthesis (1, 2, 13, 20, 21, 32). Nomura et al. (22) and Terzi (31) concluded that restrictions exerted by T-even phages on hostspecific DNA and mRNA syntheses could be divided into two classes. (i) One class is dependent on phage de novo protein synthesis and independent of multiplicity of infection (MOI). This restriction type will be referred to below as class 1. (ii) The other class is independent of phage de novo protein synthesis and dependent on MOL. This restriction type will be referred to below as class 2. In this paper we will also apply these designations to effects on host translation. Many experiments performed by several different authors (11-14, 27) have been concerned with the restriction imposed by T-even phages on inducible enzyme synthesis in Escherichia coli. Kennell (14) studied the effect of phage T4 infection on induction of fl-galactosidase (EC 3.2.1.23) in E. coli. Using the hybridization technique, he showed that lac mRNA can be induced even after T4 infection. This lac mRNA, however, was not detected in newly formed polysomes, suggesting that after T4 infection ribosomes cannot attach to the newly formed lac mRNA. Furthermore, he suggested that phage T4 blocks host translation by a mechanism independent of T4 gene expression (class 2). Rouviere et al. (27), on the other hand, 'Present address: University Institute of Microbiology, Copenhagen, Denmark.
had previously concluded from their experiments that initiation of translation of preformed host mRNA can occur after T4 infection. Kaempfer and Magasanik's (11, 12) conclusions are also in conflict with those of Kennell. They interpreted their experiments to mean that T-even phages infecting E. coli arrest the initiation of lac mRNA synthesis but do not interfere with the elongation of this mRNA. They attribute the lowered yield of enzyme obtained from preinduced cells to a twofold increase in the rate of decay of the pool of lac mRNA present at the moment of infection. They also put forward some evidence that these proposed effects of the phage infection are not dependent on phage-specific de novo protein synthesis. The present work studies the mechanism by which phage T4 interferes with inducible enzyme synthesis. We have used conditions designed to allow accumulation of a pool of lac mRNA before T4 infection and have measured its translational capacity after infection. We show that the translational yield of such a pool of lac mRNA is severely restricted in infected cells. This phageinduced restriction of host-specific translation is not dependent on phage de novo protein synthesis, occurs within 30 s after infection, and is dependent on the MOT. The restriction appears to be mediated by a mechanism preventing initiation of host-specific translation. 326
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not allow normal polysome formation, i.e., in the presence of inhibitors of protein synthesis such as CAP and puromycin (25). To obtain a large pool of preformed lac mRNA without formation of active enzyme, we therefore induced the lac operon in the presence of the amino acid analogue 7-azatryptophan, which substitutes for tryptophan in proteins (18, 23, 24, 28) and prevents formation of enzymatically active fl-galactosidase (23). 5-Methyltryptophan was simultaneously added to ensure turnoff of tryptophan biosynthesis. This analogue is not incorporated into proteins but is a potent inhibitor of tryptophan biosynthesis (3, 4, 19, 23, 24, 30). The simultaneous addition of these two analogues to induced cells completely halts the formation of active ,B-galactosidase after a short lag (Fig. 1A). This effect is reversible, as shown by the resumed increase in ,B-galactosidase activity when tryptophan is added in excess. Incorporation of [4C ]leucine is not affected (Fig. 1B), indicating that the analogues do not interfere with polypeptide synthesis, but rather cause formation of faulty proteins. Measurement of the decay of enzyme-forming capacity after RIF addition shows also that the decay of functional lac mRNA is normal (t% approximately 1.4 min) in the presence of the analogues (Fig. 2). Thus by induction with IPTG in the presence of azatryptophan and 5-methyltryptophan, it is possible to build up a steady-state pool of lac mRNA with a normal half-life without formation of active fl-galactosidase. Addition of RIF to such cells stops any further transcription. A subsequent chase of the analogues with tryptophan results in a burst of active enzyme. We will refer to this burst of active enzyme as the enzyme-forming capacity of the cells. Effect of phage T4 infection on the expression of a preformed pool of lac mRNA. The experiment summarized in Fig. 3 shows the effect of phage T4 infection on the enzymeforming capacity of a preformed pool of lac mRNA. After infection, strong inhibition (93%) of the expression of the enzyme-forming capacity occurs in infected cells (MOI, 8.8) as compared to the control (RIF-treated) culture. A trivial explanation of the observed inhibition would be that in T4-infected cells tryptophan and/or 7-azatryptophan uptake-and thus protein synthesis-is blocked. If so, this could be the cause of the observed lack of synthesis of active ,B-galactosidase in uninfected cells. The RESULTS experiment summarized in Fig. 4A and B rules lac mRNA has an abnormally high rate of out this possibility. The incorporation of ["4C]decay if accumulated under conditions that do alanine in infected cells continues in the pres-
MATERIALS AND METHODS Chemicals. Amino acids were obtained from Nutritional Biochemicals Corp., Cleveland, Ohio. ["4C lieucine (58 mCi/mmol), ["4C]alanine (171 mCi/mmol), and [benzene ring-U- 14C ]tryptophan (46 mCi/mmol) were obtained from The Radiochemical Centre, Amersham, England. Isopropyl-,8-D-thio-galactopyranoside (IPTG), onitrophenyl-,8-D-galactopyranoside, 7-azatryptophan, and 5-methyltryptophan were purchased from Sigma Chemical Co., St. Louis, Mo. Rifampin (RIF) was a generous gift from L. Carlnas, Hassle-Ciba-Geigy, Mblndal, Sweden. Chloramphenicol (CAP) was a gift from Parke, Davis & Co., Detroit, Mich. Bacteriophage. Wild-type bacteriophage T4D was provided by J. S. Wiberg. Phage T4 was grown in E. coli B, in glycerol-Casamino Acids medium (8), harvested, and purified according to the gentle method described by Goldman and Lodish (9). Phage stocks prepared according to this method showed extremely stable titers and did not contain any detectable amount of killers; i.e., the PFU values agreed with the titer estimated from the number of bacteria surviving at different MO1s. Media and growth conditions. The composition of plating agar for viable count, dilution media fo: bacteria, dilution broth for phage, and bottom and top agar for phage has been described earlier (29). All experiments were carried out in mineral salts medium M9 (7) supplemented with 0.4% glycerol. Experiments were started from cultures grown with aeration at 37 C for at least five generations to 5 x 101 to 6 x 10S cells/ml (unless otherwise stated). Sampling and assay of 0-galactosidase. At the indicated times samples of 1.0 ml were withdrawn and added to tubes with 1.0 ml of ice-cold toluene and CAP (100 Ag/ml), vigorously shaken for 15 s, and left on ice for at least 60 min. RIF was extracted by repeated toluene treatment from RIF-containing samples. The assay used is a slight modification of the method described by Miller (17). Samples of 0.5 ml were incubated at 37 C with 1.0 ml of o-nitrophenyl-,BD-galactopyranoside (0.55 mg/ml). The reaction was terminated by adding 1.0 ml of NaICO3 (1 M) and chilling on ice. Cell debris and cells were removed by centrifugation. One unit of ,-galactosidase is defined as the increase in optical density at 420 nm x 10 per min at 37 C. Measurement of protein synthesis. Samples (100 Al) of cultures labeled with a ["4C]-labeled amino acid were pipetted onto filter disks (Whatman no. 3) at the indicated times, and 15 s later the disks were dropped into ice-cold 5% trichloroacetic acid and left in cold trichloroacetic acid for at least 60 min. They were then washed twice with cold trichloroacetic acid and boiled for 1 h in trichloroacetic acid. Finally, they were washed three times with cold trichloroacetic acid and dried. Radioactivity was determined in a toluenebased scintillation mixture.
328
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FIG. 1. (A) Effect of 7-azatryptophan (7-AT) and 5-methyltryptophan (5-MT) onfI-galactosidase formation. To an exponentially growing culture of E. coli B (5.3 x 108 cells/ml), IPTG (5 x 10J 4 M) was added at time zero. To a part of the culture, 7-AT (50 ig/ml) and 5-MT (5 igg/ml) were added at 8 min. The latter culture was again divided, and tryptophan (1,250 Ag/ml) was added to one part at 16 min. fl-Galactosidase activities in: (a) control culture (0); (b) culture with 7-AT and 5-MT (0); (c) culture with tryptophan added after 7-AT and 5-MT (V). (B) Protein synthesis during treatment with 7-AT and 5-MT. Experimental conditions were identical to those described in (A), except that ["CC]leucine (0.4 gtCi/ml, 2.4 4g/ml) was added at I min. Samples were withdrawn to trichloroacetic acid at the indicated times. Hot trichloroacetic acid-insoluble radioactivity in: cells before addition of analogues (0); cells treated with 7-AT and 5-MT (0); cells with tryptophan added after 7-AT and 5-MT (V)-
of the tryptophan analogues (Fig. 4A). The rate of incorporation is not increased upon a chase of the tryptophan analogues with excess tryptophan, suggesting that the translational rate in infected cells is not affected by the presence of the analogues. Furthermore, analogue-treated infected cells incorporate exogenously supplied tryptophan into proteins (Fig. 4B). These experiments show that the lack of synthesis of active fl-galactosidase after infection is not due to any deficiency in uptake or rate of incorporation into proteins of exogenously supplied tryptophan. Phage T4 restricts host-specific translation independently of phage genome expression. The immediacy of the observed inhibition suggests that it is independent of de novo expression of phage genes. To test this ence
assumption we added RIF before T4 infection of the preinduced cells. Restriction of the enzymeforming capacity equivalent to that in the absence of RIF was found (Fig. 5). When tryptophan is added 30 s after phage infection the yield of fl-galactosidas6 is 90% less than in uninfected cells. Thus, T4 blocks the expression of preformed lac mRNA also in the absence of phage-induced RNA synthesis. This conclusion rests upon the assumption that the concentration of RIF used (200 Ag/ml) was adequate to block T4 gene expression. The following experiment was performed to test this assumption. Different concentrations of RIF were added to uninfected E. coli 1 min before induction with IPTG. At a concentration of 200 jug/ml, no rise in f3-galactosidase activity could be detected (Fig. 6). If complete inhibition of
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lysis from without. Lysis from without does not normally occur at a moderate MOI, but could conceivably occur at a lower MOI when T4 gene expression is prevented. Since RIF treatment stops transcription irreversibly, measurement of infective centers or T4 gene functions could not be used to exclude that significant lysis from without had occurred. We therefore performed an experiment based on the following argument. fl-Galactosidase is an intracellular enzyme, which is not released from the cells into the surrounding medium. Thus, by a wave of induction, a pool of f-galactosidase can be accumulated within the cells. If T4 infection
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transcription for one given mRNA can be taken as evidence that all RNA polymerase molecules are blocked, this experiment rules out the possibility that T4 restriction of enzyme-forming capacity is caused by de novo expression of a bacteriophage gene. MOI dependence of restriction of hostspecific translation. As the observed phageinduced restriction does not depend on de novo expression of phage genes, we investigated whether the restriction was dependent on the MOI. In uninfected cells the preformed lac mRNA determines the formation of a certain amount of active enzyme upon addition of tryptophan. In infected cells only a fraction of this enzyme-forming capacity is expressed. Figure 7 shows that the logarithm of the fraction of the enzyme-forming capacity decreases linearly with increasing MOI. Phage T4-induced restriction of hostspecific translation is not due to lysis from without. It could be argued that the observed T4-induced inhibition of fl-galactosidase formation from pre-existing lac mRNA in the presence
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FIG. 3. Phage T4-induced restriction of translation of preformed lac mRNA. An exponentially growing culture of E. coli B (5.8 x 108 cells/ml) was divided. To one portion the following additions were made: 7-azatryptophan (7-AT) (50 lg/ml) and 5-methyltryptophan (5-MT) (5 ;g/ml) at -4 min and IPTG (5 10-' M) at 0 min. The induced culture was then subdivided into two aliquots. At 5.5 min phage T4 (MOI 8.8) was added to one aliquot and RIF (200 ;ig/ml) was added to the other, and at 7 min tryptophan (1,250 jig/ml) was added to both. To the uninduced portion of the original culture CAP (50 jug/ml) was added at -3 min to avoid increase in background activity due to increase in cell concentration. ,8-Galactosidase in: (A) control culture with CAP added at -3 min (0); (B) culture induced at 0 min in the presence of 7-AT and 5-MT (0); (C) same as in (B) but with RIF added at 5.5 min and tryptophan at 7 min (V); (D) same as in B but with T4 (instead of RIP) added at 5.5 min and tr-yptophan at 7 min (V). x
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MINUTES AFTER INFECTION MINUTES AFTER INFECTION FIG. 4. (A) Protein synthesis in T4-infected cells during treatment with 7-azatryptophan (7-AT) and 5methyltryptophan (5-MT). To an exponentially growing culture of E. coli B (4.5 x 108 cells/ml), 7-AT (50 pg/ml) and 5-MT (5 gig/ml) were added at -3 min. At zero time the culture was infected with T4 (MOI 5.6), and at I min [14C]alanine (0.4 gCi/ml, 2.5 uig/ml) was added. To a part of the infected culture tryptophan (1,250 Ag/ml) was added at 9 min. Samples were withdrawn to trichloroacetic acid at the indicated times. Hot trichloroacetic acid-insoluble radioactivity in: 7-AT- and 5-MT-treated cells (0); 7-AT- and 5-MT-treated cells after addition of tryptophan (0). (B) [14C]tryptophan incorporation in T4-infected cells during treatment with 7-AT and 5-MT. To an exponentially growing culture of E. coli B (5.5 x 108 cells/ml) 7-AT (50 ,g/ml) and 5-MT (5 POg/ml) were added at -3 min. At zero time the culture was infected with T4 (MOI 4.1), and at 1 min [14Cjtryptophan (0.23 ,gC'i/ml, 0.47 Aglml) was added. Samples were withdrawn to trichloroacetic acid at the indicated times.
of such cells leads to lysis from without, the intracellular pool of fB-galactosidase should be released into the surrounding medium. If, on the other hand, no lysis from without occurs, the pool of fi-galactosidase should be withheld by the cells. Table 1 shows the results of such an experiment. No detectable 3-galactosidase activity is released from cells even at a high MOI. After T4 infection at high multiplicites, the /3galactosidase activity in cells was slightly reduced. Since no corresponding amount of free /3-galactosidase was found in the medium, the decrease is probably not an indication of lysis from without. At any rate, the decrease in the detected intracellular 0-galactosidase activity (9% at MOI 8.5) is too small to account for the almost total restriction (91%) of lac mRNA translation seen at this MOI (Fig. 4). We also interpret the findings of Nomura et
al. (22) as evidence against lysis from without in the absence of protein synthesis. They infected CAP-treated cells with T4 at multiplicities two times higher than in our experiments without loss of infective centers.
Kinetics of T4-induced inhibition of f3-galactosidase formation. The course of fl-galactosidase formation from preformed mRNA after T4 infection (Fig. 3 and 5) suggests that the initiation of translation of the lac mRNA might be specifically inhibited. To test this hypothesis we performed the following experiment. Cells were induced with IPTG at time zero, and samples were withdrawn at different times into tubes containing either CAP or T4. The tubes with T4-infected samples were incubated for 30 min after sampling to allow complete expression of the lac mRNA. The final enzyme activity attained in each tube is plotted versus
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the hypothesis that T4 interferes with the initiation of host-specific translation.
5 10 15 20 25 MINUTES AFTER INDUCTION
FIG. 5. Phage T4-induced restriction of translation of preformed lac mRNA in the presence of RIF. An exponentially growing culture of E. coli B (4.4 x 10' cells/ml) was divided. To one portion the following additions were made: 7-azatryptophan (7-AT) (50 yg/ml) and 5-methyltryptophan (5-M7T) (5 ,gg/ml) at -4 min, IPTG (5 x 10-4 M) at 0 min, and RIF (200 ,pg/ml) at 5 min. This induced culture was then subdivided into two portions; at 6 min one aliquot was infected with phage T4 (MOI 8.6) and the other was sham-infected; at 6.5 min tryptophan (1,250 $&g/ml) was added to both. To the uninduced portion of the original culture, CAP (50 jg/ml) was added at -3 min to avoid increase in background activity due to increase in cell concentration. P-Galactosidase in: (A) control culture with CAP added at -3 min (0); (B) culture induced at 0 min in the presence of 7-AT and 5-MT and with RIF added at 5 min (0); (C) same as in (B) but with tryptophan added at 6.5 min (v); (D) same as in (C) but with T4 added at 6 min (v). the time of sampling in Fig. 8. If phage infection interferes with initiation of translation and not with elongation the following predictions can be made. (i) The T4 curve should start to rise from time zero. (ii) When steady-state conditions of transcription and translation are reached, the values on the T4 curve should exceed those on the CAP curve with a constant value (representing the capacity of ribosomes already in progress to produce
B-galactosidase polypeptide chains). (iii) The time difference between the parallel parts of the two curves should be one z gene translation time (estimated in our system to be approximately 1.6 to 1.7 min). Figure 8 shows that all these predictions are fulfilled, a result that supports
DISCUSSION During the last decade a great number of experimental data concerning the mechanism by which T-even phages shut off host macromolecular synthesis have accumulated. For reviews, see Mathews (16) and Duckworth (5). Several different hypotheses have been put forward to explain the restriction of inducible enzyme synthesis in E. coli upon T-even phage infection; namely, the phage (i) restricts initiation of host mRNA transcription (12), (ii) causes a twofold increase in host mRNA decay (11), and (iii) interferes with the translation of host mRNA (14). We find that a mechanism according to hypothesis (iii) can account for the restriction of host protein synthesis at multiplicities normally used in phage experiments. In uninfected cells lac mRNA was shown to accumulate upon induction in the presence of
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MULTIPLICITY OF INFECTION FIG. 7. Dependence on MOI of the T4-induced restriction of translation of preformed lac mRNA. To an exponentially growing culture of E. coli B (5.4 x 106 cells/ml) were added: 7-azatryptophan (7-AT) (50 sgg/ml) and 5-methyltryptophan (5-M7) (5 ig/ml) at time zero, IPTG (5 x 10-4 M) at 4 min, and RIF (200 jlg/ml) at 9 min. The culture was divided into six aliquots. To each aliquot, phage T4 was added at 10 min and tryptophan (1,250 ,ug/ml) was added at 11 min. Incubation was continued for 34 min. fl-Galactosidase activity was measured and corrected for the activity in uninduced cells (1.9 units), and at each MOI, for the calculated enzyme formed by uninfected cells. The activity was expressed as percentage of the value (11.25 units) in uninfected cells. TABLE 1. Distribution of preformed fl-galactosidase between cells and culture medium after T4 infection
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a To an exponentially growing culture of E. coli B (5.0 x 101 cells/ml), IPTG (5 x 10-4 M) was added at time zero and RIF (200 ,g/ml) was added at 2 min. The culture was divided into aliquots. Each aliquot was infected with phage T4 (at the indicated multiplicity) at 12 min. From each aliquot a sample (4.0 ml) was withdrawn and centrifuged for 10 min at 12,000 x g. j%-Galactosidase was determined in the supernatant and pellet fraction after resuspending it in 4.0 ml of fresh medium. As a control of the total ,B-galactosidase activity another sample (2.0 ml) was withdrawn from each aliquot at 16 min and assayed for fl-galactosidase without prior centrifugation.
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7-azatryptophan, a significant burst of active being formed after addition of tryptophan in the presence of RIF (Fig. 1-3, 5). The burst of enzyme is strongly suppressed upon phage T4-infection (Fig. 3). This result rules out the possibility that the observed restriction of host enzyme synthesis takes place at the step of transcriptional initiation (hypothesis [i]). We conclude that phage T4 interferes with the translation of the preformed mRNA. A twofold increase in the rate of lac mRNA degradation (hypothesis [ii]) is insufficient to explain the considerable reduction of enzyme yield after infection (Fig. 3 and 5). Phage T4 efficiently prevents expression of enzyme
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preformed lac mRNA even in the presence of RIF. Since RIF blocks RNA polymerase in both infected and uninfected cells (10, 33), this experiment shows that the T4-induced restriction of host translation is not dependent on phage de novo protein synthesis (class 2 type). The class 2-type effects on host mRNA and DNA synthesis described by Nomura et al. are characterized by being MOI dependent. Also the T4-induced restriction of translation of host mRNA, which we describe, is MOI dependent (Fig. 7). Our results are in accordance in all respects with the findings and interpretations of Kennell, but they are at variance with some of the interpretations put forward in papers by Kaempfer and Magasanik (11, 12) and Rouviere et al. (27). However, the present results and the findings by all of these authors are in agreement if it is assumed, as suggested by Kennell, that T-even phage infection interferes with new translational initiating events on host mRNA. Our measurement of the yield of f-galactosidase after different induction periods before infection (Fig. 8) argues that T4 interferes with host protein synthesis by inhibiting initiation of translation. There is an apparent discrepancy between this hypothesis and an experiment by Rouviere et al. (27), which they interpreted as evidence for continued initiation of translation of the preformed host mRNA after T4 infection. They used p-fluorophenylalanine incorporation to produce a heat-labile form of fl-galactosidase. Cells grown in medium containing p-fluorophenylalanine were induced with IPTG and then infected with T4. Thereafter, aliquots of the induced cells were withdrawn and diluted into medium containing phenylalanine. By this technique, induction was stopped by dilution of inducer and phenylalanine was permitted to enter into the C-terminal portions of the flgalactosidase-specific peptides from the time of dilution. Using this system Rouviere et al. could rescue heat-stable ,-galactosidase, although at rapidly decreasing levels, as late as 2 min after infection. This result led them to conclude that new translational initiations on host mRNA occur after T4 infection. Previous work by Kepes (15) shows that incorporation of p-fluorophenylalanine in small C-terminal portions makes fl-galactosidase heat labile (15) (Fig. 5). However, when cells were first grown in the presence of p-fluorophenylalanine and then given phenylalanine, i.e., incorporation of the analogue in the main portion of the N-terminal part and phenylalanine in a small C-terminal
part of the peptide, the enzyme became almost as heat stable as normal enzyme. Incorporation of phenylalanine in a very small part of the C-terminal end of such an analogue-containing enzyme is needed to make it heat stable. This is shown by the fact that 75 to 80% of the heat-stable enzyme was found when phenylalanine was added as late as 2 min after translation in the presence of p-fluorophenylalanine, i.e., approximately one translation time for the z gene. Values are calculated from Kepes (15) (Fig. 6). Hence, the heat-stable enzyme found by Rouviere et al. upon addition of phenylalanine after phage infection may be composed of peptide chains initiated before infection but completed in the presence of phenylalanine, i.e., containing phenylalanine in their C-terminal portions and thereby rendering them heat stable. The observed time limit for rescue of heat-stable enzyme agrees with the translation time of the z gene. Thus, also in this respect, their results are in accordance with our findings. In short, we believe that there is agreement between the results obtained by others and the hypothesis suggested by Kennell and us that phage T4 interferes with the initiation of translation of host mRNA. Several mechanisms for this inhibition have been suggested. In particular, Kennell (12) discussed thoroughly what cellular structure might be the target of the phage action. Our observations argue for an explanation based on some modification of intracellular conditions that are a consequence of attachment and/or penetration. Thus we find that the inhibition of fl-galactosidase synthesis is dependent on the MOT. Furthermore, it is independent of phage protein synthesis after infection, also occurring during infection in the presence of RIF. The formal similarity between T4 phage infection when transcription is blocked and T4 ghost infection raises the possibility that inhibition of ,B-galactosidase synthesis caused by T4 infection and that caused by ghost infection have the same mechanism. However, the fact that ghost infection strongly interferes with simultaneous phage infection (6, 26) indicates that ghost infection has a different and more radical effect on the translational machinery. We are investigating the effects of ghost on ,B-galactosidase induction in some detail (un-
published data). Whatever the nature of the change that dramatically curtails the initiation of translation from lac mRNA in T4-infected cells, we want to focus attention on its specificity; it does
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