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Regulation of the synthesis of bacteriophage T4 gene 32 protein
J. Mol. Biol. (1974) 88, 89-104
Regulation of the Synthesis of Bacteriophage T4 Gene 32 Protein H. M. KRISCH,
A. BOLI,E
AND R. H. EPSTEIN
Dipartement de Biologic MolCculaire Universitk de Gentve, Geneva, Switzerland (Received 20 February 1974) The synthesis of T4 gene 32 product (P32) has been followed by gel electrophoresis of infected cell lysates. In wild-type infections, its synthesis starts soon after infection and begins to diminish about the time late gene -expression commences. The absence of functional P32 results in a marked increase in the amount of the non-functional P32 synthesized. For example, infections of T4 mutants which contain a nonsense mutation in gene 32 produce the nonsense fragment at more than ten times the maximum rate of synthesis of the gene product observed in wild-type infections. All of the temperature-sensitive mutants in gene 32 that were tested also overproduce this product at the non-permissive temperature. This increased synthesis of the non-functional product is recessive, since mixed infections (wild-type, gene 32 nonsense mutant) fail to overproduce the nonsense fragment. Mutations in genes required for late gene expression (genes 33 and 55) as well as some genes required for normal DNA synthesis also result in increased production of P32. The overproduction in such infections is dependent on DNA synthesis; in the absence of DNA synthesis no overproduction occurs. This contrasts with the overproduction resulting from the absence of functional P32 which is not depeadent on DNA synthesis. These results are compatible with a model for the regulation of expression of gene 32 in which the synthesis of P32 is either directly or indirectly controlled by it& own function. Thus,. in the absence of ,P32 function the expression of this gene is increased as is manifest by the high rate of P32 synthesis. It is further suggested that in infections defective in late gene expression and consequently of replicated DNA, the increased P32 production is caused in the maturation by the large expansion of the DNA pool. This DNA is presumed to compete for active P32 by binding it non-specifically to single-stranded regions, thus reducing the amount of P32 free to block gene 32 expression. Similarly, the aberrant DNA synthesized following infections with mutants in genes 41,56,58,60 and 30, although quantitatively less than that produced in the maturation defective infections, can probably bind large quantities of P32 to single-stranded regions resulting in increased P32 synthesis.
The
product
DNA synthesis Amber mutants
of bacteriophage (Epstein in this
1. Introduction T4D gene 32 (P32)
is required
et aZ., 1963 ; Warner & Hobbs, gene fail
to undergo
more
than
continuously
for
T4
1967 ; Alberts & Frey, 1970). a single round of replication
in the non-permissive host (Kozinski & Felgenhauer, 1967) and a temperaturesensitive mutant ceases DNA repliciltion within a minute after shifting to the nonpermissive temperature (Riva et al., 1970a). Gene 32 appears to be unusual among 89
H.
90
M. IIRISCH,
A. BOLLE
AND
R. H.
EPSTEIN
the DNA synthesis genes in that its product is required in very large amounts (104 molecules/cell) for normal replication (Alberts & Frey, 1970). Furthermore, the level of P32 may control the amount of DNA synthesis (Sinha & Snustad, 1971). P32 binds co-operatively t)o single-stranded DNA, and facilitates the denaturation and renaturation of DNA under physiological conditions (Alberts & Frey, 1970). It has is that of stabilization of the singlebeen suggested that the role of P32 in replication stranded regions which exist transiently near the growing point of a replicating DNA molecule. Such regions presumably present an efficient template for the replication machinery (Alberts & Frey, 1970; Delius et al., 1972). The data presented in this paper suggest that gene 32 protein has a role in the regulation of its own synthesis. The absence of functional gene 32 product results in a marked increase in the amount of defective P32 synthesized. For example, in infections with T4 strains containing a nonsense mutation in gene 32, the nonsense fragments are made at ten times the maximum rate of synthesis of the gene 32 product in a wild-type infection. In addition, evidence will be presented that, because of the properties of P32, the nature and amount of DNA replication in an infected cell also have an effect on the expression of gene 32.
2. Materials and Methods (a) Bacterial strains Escherichia coli BE (sup”) from the Geneva collection was used as the host bacterium in all experiments. E. coli CR63 (sup’ 1) was used to prepare phage stocks and as the permissive host for phage containing amber mutations. (b) Bacteriophage All of the phage used in these experiments are mutants of’ T4D. The wild-type T4D and the r1 mutant, r45, used in this laboratory were originally obtained from A. H. Doermann. Except for the gene t mutant, tamA (Josslin, 1970,1971), the amber and temperature-sensitive mutants are described in Epstein et al. (1963). Phage containing combinations of these mutations were constructed by standard cross procedures (Krisch, et cd., 1972). The identification of complex genotypes was accomplished following the methods described by Doermann & Boehner (1970). The following mutant phages were used: rIr48, 3OamH39X, 32tsP7, 32tsL94, 32tsLl70, 32tsG26, 32amA453, 32amH18, 32amHL618,4ltsCB115,41amN81,42tsLB3,42aniN122,43tsP36,43amB22, 43amB263,44am.N82, 45tsL159, 45amE10, 46amN130, 55amBL292, BGamEABl, 58arnYlZA219, 60amHL626, 2lamN90, 22amE209, 23amHl1, 23amB17, eamH26, and IP” (internal protein defective, Showe & Black, 1973). (c) Media M9S
contains 0.2% Casamino acids was of host bacteria. 1962) was used as the growth medium for the broth (Steinberg & Edgar, of indicator bacteria. Phage and bacteria were plated on Hershey bot.tom
medium
(Champe
used in the preparation Hershey
preparation agar using EHA
& Benzer,
1962)
which
of stocks and for the growth
(Steinberg
& Edgar, 1962) top layer agar. (d) Experimental
Host
cells
were
grown
procedure
at 37°C to 5 X 107/ml
in M9S medium
and then
centrifuged
and resuspended at a cell density of 1.4 X 10s in M9S.l which contains only O*OZ”h Casamino acids. Unless otherwise indicated, the entire , a,t 30%. Exponential E. coli BE cells at a density added to the adsorption tube with aeration 2 min greater vol.--of M9S. 1 medium containing phage at
subsequent procedure was carried out of 1.4~ luQ/ml in M9S.l medium were before the addition of phage. A sixfold a concn of-2.3 x 10Q/ml (a multiplidity
REGULATION
OF T4 GENX
91
3.2 PROTEIN
of 10) was added to the absorption tube to begin the infection. Adsorption under these conditions was rapid and essentially all cells were infected by 3 min after the addition of phage. At the times indicated in the text and Figure legends, 5 ml of infected cells (2 x lO*/ml) were added to 0.5 ml of a mixture of 15 14C-labelled amino acids (New England Nuclear, NEC 445) at 10 ~Ci/ml. Four minutes later, 0.5 ml of 20% Casamino acids was added to terminate the labelling and after a 5-min chase, the samples were put in ice. (e) Radioactive
lysate preparation, gel electrophoresis of protein yields determination
arbd
The methods were identical to those previously used in this laboratory by Russcl (1973). The determination of relative protein yield was accomplished by exposing X-ray film (Kodak) to the dried gel slices or slab for a period of 1 to 6 days. The autoradiographs were developed and then traced with a Joyce-Loebl recording microdensitometer. The area of the peak corresponding to the protein of interest was determined by cutting out the peak and weighing it. The amount of trichloroacetic acid-precipitable counts in each lysate was determined and in general this was similar for different lysates within a single experiment. The data was normalized by dividing the peak area by the number of trichloroacetic acid-precipitable counts added to the gel. The value of the peak area/&s per min was taken as the average rate of synthesis during the period of the pulse labelling. All values shown in a given Figure (or Table) were determined using lysates from the same experiments, analysed in the same gel. The values obtained in separate experiments
have not been standardized for exposure time etc; consequently, the numerical values in different Figures are not to be compared. To compare the relative yield of the whole P32 protein and the fragment produced in 32amH18 infections, it was necessary to correct the band intensity measurements for the difference in molecular weight of the two products (P32=35,000; 32amH18= 17,000). Molecular weights were estimated from gel migration rates relative to phage T4 early proteins, the molecular weights of which had been determined (O’Farrell et al., 1973). All of the gels from which quantitative data were taken were 10% gels in which the P32 band and the adjacent PrIIBt band were well separated. A discussion of the sensitivity and sources of error in measurements of band intensities on gels is to be found infection (4 to 8 min) the label in the P32 in Russel (1973). Early in a lysis-defective band represents about 10% of the labelled protein in the gel.
3. Results Following appearance
the entry
of the phage T4 genome into a host cell, there is an ordered
of phage-specific
protein
(Hosoda
& Levinthal,
1968). Almost
immediately
after infection a group of enzymes are synthesized which are involved in the replication of phage DNA. Synthesis of these enzymes proceeds for 10 to 20 minutes at 30°C and then essentially stops (Wiberg et al., 1962). The control mechanism underlying this cessation of early enzyme synthesis is poorly understood; however, studies with mutants unable to synthesize phage DNA demonstrate that normal turn-off requires phage DNA synthesis (Wiberg et al., 1962). The sequence of gene expression in T4-infected cells can now be studied in considerable detail by the analysis of newly synthesized proteins pulse-labelled with l 4C-labelled amino acids. After a chase with unlabelled amino acids, the infected cells are lysed and a sample of the crude lysate analysed by electrophoresis on a sodium dodecyl sulphate-polyacrylamide slab gel. Autoradiographs of such gels make it possible to identify many of the T4specific proteins. Tracing of the autoradiographs with a microdensitometer permits t Abbreviations used: PrIIB, the protein product of wild-type phage T4 gene TIIB; phage mutant in such genes synthesize little if any DNA (Warner & Hobbs, 1967).
DO gene,
92
H.
M. KRISCH,
A. BOLLE
AND
R. H.
EPSTEIN
the measurement of the relative amounts of various proteins synthesised during the labelling period. The product of T4 gene 32 can be identified easily on sodium dodecyl sulphatepolyacrylamide gels. Plate I shows that lysates from infections with phage carrying any one of three different amber mutations in this gene are missing a single band which migrates with a molecular weight of 35,000. This estimate of the molecular weight of P32 is in good agreement with the value determined by Alberts & Frey (1970). In addition, each mutant lysate contains a single new band which corresponds to the nonsense fragment. The 32amH18 fragment has a molecular weight of 17,000, that of 32amHL618 is about 10,000 and the 32amA453 fragment is yet smaller. Identification of P32 on sodium dodecyl sulphate-polyacrylamide gels makes it possible to determine the time course of its synthesis by pulse-labelling at various times during infection and determining the amount of this protein synthesized during each pulse. Figure 1 shows the pattern of P32 synthesis in a lysis-defective infection. (At comparable times the patterns of P32 synthesis in wild-type and tamA (lysis-defective) infections are identical.) The synthesis of P32 commences early in infection and continues at a high rate until after the initiation of replication and of late gene expression. About 15 minutes after infection at 3O”C, P32 synthesis begins to diminish; however, unlike many other pre-replicative proteins, its synthesis is still significant long after the initiation of replication.
Time
after
mfectmn
(msn)
FIG. 1. Rates of synthesis of P32 in gene t (lysis-defective), gene 44, and gene 32 mutantinfected cells. E. c&i BE at 2 x 108/ml were infected at 3O’C with tamA ( l ), 44amN82 (m), or 32amH18 (A) at a m.o.i. of 10 and labelled at various times with 14C-labelled amino acids. Each labelling was followed by a 6-min chase with unlabelled amino acids. m.o.i., multiplicity of infection. Each point in the Figure represents the average rate of synthesis of P32 during a 4-min pulselabelling. Rates are plotted at the midpoint of each pulse interval. See Materials and Methods for additional experimental details.
When DNA synthesis is stringently blocked by a mutation in gene 44 (Warner & Hobbs, 1967), the shut-off of many early products, including P32 does not occur. Such an infection (Fig. 1) results in an extended period of P32 synthesis at nearly the maximum rate observed in a wild-type infection. A number of T4 mutants completely blocked in DNA synthesis (mutants in the DO genes: 42, 43, 44, 45) result in a similar pattern of P32 synthesis with the notable
amXS
amHI
amHl.618
ICE Y --------u-----r-----------------------
h cd Y
1
REGULATION
OF T4 GENE
32 PROTEIN
93
exception of mutants in gene 32 itself. In infections with gene 32 mutants, the absence of functional P32 results in a marked increase in the amount of the nonfunctional product synthesized. As can be seen from Figure 1, the synthesis of the defective product (nonsense fragment) occurs at a continually increasing rate throughout the course of infection. At 38 minutes after infection at 30°C with a gene 32 nonsense mutant (32amH18) the rate of synthesis of the defective product is 40 times that observed at a comparable time in the lysis-defective infection. The increased rate of P32 synthesis relative to wild-type rate is hereafter called P32 overproduction. In order to examine in more detail the synthesis of P32 in the absence of its function a series of pulse-labellings were carried out using T4 strains containing nonsense mutations in gene 32 or gene 43 (a DO gene) as well as with a strain which contains mutations in both genes. As was demonstrated in Figure 1 with a mutant in a different DNA synthesis gene (gene 44), the failure to replicate DNA results in an extended period of P32 synthesis. The data in Figure 2 demonstrates that the rate
'0
t 5 Time
1 I I I I IO 15 20 25 30 after
infection
(min)
FIG. 2. Rates of synthesis of P32 in gene 43, gene 43-gene 32, and gene 32 mutant-infected cells. E. COG BE at 2x lO’/ml were infected at 30°C with 43amB22 (a), 43amB22-32amH18 (m), of 32amH18 ( A) at a m.o.i. of about 10. At the indicated times, the infected cells were labelled with l*C-labelled amino acids for 4 min, followed by a 5-min chase with unlabelled amino acids. Rates are plotted as described in the legend to Fig. 1.
of synthesis of non-functional P32 is approximately ten times that observed in a comparable infection in which DNA synthesis is blocked by a mutation in gene 43 (43amB22). The possibility that the small amount of DNA synthesized in the 32amH18 infection (Kozinski & Felgenhauer, 1967) contributes to the overproduction of P32 has been excluded by constructing the double mutant 43amB22-32amH18 and testing it for P32 overproduction (Pig. 2). The results of this experiment demonstrate that P32 overproduction by a gene 32 mutant is not affected by a complete block in DNA synthesis.
94
H.
M. KRISCH,
A. BOLLE
AND
R. H.
EPSTEIN
TABLE 1 Gene 32 product yield relative to wild type after infection with temperature-sennsitive gene 32 mutants
t See Materials
Mutation
Relative yield? 15 to 19 min after infection
32tsp7 32tJsL94 32kL170 32tsG26
6.3 6.2 6.4 3.9
and Methods
for additional
experimental
at 42°C
details.
The data in Table 1 indicate that four temperature-sensitive mutations in gene 32 also result in increased synthesis of P32 at the non-permissive temperature. This increased P32 synthesis cannot be attributed merely to the block in DNA replication in such infections, since additional experiments indicate that the rate of P32 synthesis (in a 15 to 19 min pulse at 42°C) is at least five times greater in a 32tsP7 infection than in a 43tsP36 infection. The mutant 32tsG26, which synthesizes less P32 than the other temperature-sensitive mutants, is the leakiest of the gene 32 mutants in that, unlike the others, it makes minute plaques (on CR63) with a high efficiency at the non-permissive temperature.
4. Self-regulation The results in the previous sections indicate that P32 is either directly or indirectly involved in the regulation of its own synthesis. It has been observed previously in this laboratory that the expression of T4 gene 43 (DNA polymerase) is also regulated by its product (Russel, 1973). In view of these results it was considered important to establish whether this type of regulation was a common feature of the gene products involved in phage DNA synthesis. Experiments have been carried out at the non-permissive temperature with temperature-sensitive mutants in some of the other DNA synthesis genes. These experiments indicate that the products of gene 4I (tsCBllti), gene 42 (tsLB3), and gene 45 (tsL159) are not overproduced in the absence of their function (data not shown). These observations suggest that self-regulation is not common to all of the DNA synthesis genes but is restricted to only some of them. It has not been rigorously excluded that there are yet more self-regulatory genes in T4 (involved either in DNA synthesis or in other aspects of the life cycle). This question is currently under more extensive investigation.
5. Overproduction The relation was examined The results of sense fragment
: Recessiveness of the Gene 32 Mutant Allele
between the amount of functional P32 and the level of P32 synthesis in a series of mixed infections with wild-type and gene 32 mutants. these experiments demonstrate that the overproduction of P32 nonis recessive to the wild-type allele for gene 32. In the experiment
REGULATION
OF T4 GENE
32 PROTEIN
95
shown in Plate II(a), a mixed infection of T4D+ and 32amHl8 was carried out with equal multiplicities of each type, and the amount of P32 and the 32amH18 fragment synthesized during a pulse was determined. As can be seen from Plate II(a), the presence of functional P32 greatly reduces the rate of synthesis of the amber fragment. The interpretation of this experiment is complicated by the fact that in both the wild-type control and in the mixed infection, DNA synthesis and late gene expression occur. In contrast in the other control experiment, a 32amH18 infection, neither DNA synthesis nor expression of late genes takes place. Consequently a direct comparison of the three infections is difficult. Since this complication can be circumvented by using a nonsense mutant in gene 43 instead of wild type as a control, the mixed infection was carried out with the 43amB22 strain and a strain which has mutations in both gene 43 and 32 (43amB22-32amH18). These infections (Plate II(b)) confirm the previous conclusion that functional P32 greatly reduces the rate of synthesis of the non-functional P32 fragment. In the experiment shown in Figure 3, cells were infected at the same time with different ratios of amber and wild-type phage, and the amount of P32 was measured. It is interesting to note that in the mixed infection the synthesis of P32 is the same as in the infeotion with wild type alone. In addition it is clear from the quantitative analysis of these experiments (as well as the experiments shown in Plate II) that the nonsense fragment and the intact P32 are produced in direct proportion to the input ratio.
FIG. 3. Rates of synthesis of wild-type P32 in mixed infections with wild-type and gene 32 amber mutant. E. coZi BE at 2.0 x lOs/ml were infected at 30°C with wild type alone (a), wild type and 32amH18 at an input ratio of 2 to 1 ( n ), or wild type and 32amH18 at an input ratio of 1 to 2 (A). Total multiplicity of infection in all cases was 15. At the indicated times, the infected cells were labelled with 14C-labelled amino acids for 4 min, followed by a 5-min chase with unlabelled amino acids. Rates are plott,ed as described in the legend to Fig. 1.
6. Continual Requirement of Gene 32 product for the Regulation of its Synthesis Since there is a continuous requirement of P32 for DNA synthesis, it may be asked whether the level of functional P32 regulates its own syntheGs throughout the infectious cycle. The experiment shown in Figure 4 involves an infection of E. coli BE at 30°C with a temperature-sensitive mutation in gene 32 (32tsP7), and a parallel control with a temperature-sensitive gene 43 mutant (43tsP36). After the synthesis of P32 began to diminish at 30°C the infected cells were shifted to 42~5°C. Both of the temperature-sensitive-mutant products are rapidly inactivated at the
96
H.
hf. KRISCH,
A. BOLLE
AND
R. H.
EPSTEIN
4( Time ofter infection (mini FIG. 4. Rates of synthesis of P32 before and after a shift from 30°C to 42.5’C at 22 min after infection. E. coli BE at 2 x lOs/ml were infected with 43tsP36 or 32tsP7 at a m.o.i. of 10 at 30°C. At the indicated times, portions of the culture were pulse-labelled for 4 min and then chase’d for 5 min with unlabelled amino acids. At 22 min after infection a portion of the culture was diluted with an equal volume of medium at 54’C and placed at 42.5”C. Following the temperature shift two pulse-labellings were carried out on the portion of the culture at 42.5’C. Rates are plotted as described in the legend to Fig. 1; (0) 43tsP36; (a) 32tsP7. The arrow ( 4 ) indicates the time of shift; the broken line indicates that portion of the culture shifted to 42.5% while the solid line was that portion maintained at 30°C throughout the experiment.
non-permissive temperature since DNA replication ceases within a few minutes (Riva et al., 1970a). The data in Figure 4 demonstrate that the temperature shift which renders the thermosensitive P32 inactive for replication also renders it nonfunctional for regulation of its own synthesis. Soon after the temperature shift P32 synthesis increases nearly fivefold. In the same experiment carried out with the temperature-sensitive gene 43 mutant, the increase in P32 synthesis is only about twofold. Since the temperature shift was carried out at 22 minutes, these results also indicate that the infected cell maintains the capacity to augment the synthesis of P32 in response to a deficit of the functional product well into the late period. It should be noted that even at the permissive temperature 32tsP7 synthesizes nearly twice the normal complement of P32. A possible explanation for this is thaf the 32tsP7 protein is only partially functional at the permissive temperature and twice the normal amount is required to attain the wild-type level of P32 function.
7. Role of DNA Synthesis in Gene 32 Regulation The amount of functional P32 appears to determine both the level of DNA synthesis and also the rates of its own synthesis. The nature of the relationship between DNA synthesis and the expression of gene 32 have beed investigated in a number of experiments. As was demonstrated in the experiments shown in Figures 1 and 2 infections with mutants in genes 43 or 44 (with less than 1% of the wild-type level of DNA synthesis (Warner t Hobbs, 1967)) result in an extended period of P32 gene expression. An identical pattern of P32 synthesis is obtained when DNA synthesis is gtringently blocked by use of a phage mutant. in genes 41 to 45 (see Fig. S).. As is shown in Figures 5 and 6 a number of mutants which are not rigorously blocked in DNA synthesis (Warner t Hobbs, 1967) have a quite different pattern of P32 synthesis. Mutants which synthesize a significant amount of DNA (gene 41,
REGULATION
OF T4 GENE
33
A’
li,
.-* .-*
32 PROTEIN
97
56-“A
,i
IO iime
15 20 25 30 after infection (mini (min)
FIG. 6. Rates of synthesis of P32 in mutants with aberrant DNA synthesis. E. coli BE at 2.0~ lOs/ml were infected at 30°C with T4Dr48 (a), 41amN81 ( n ), or 56amEA51 (A) at a m.o.i. of about 10. At the indicated times the infected cells were labelled with 14C-labelled Ltmino acids for 4 min, followed by a 6-min chase with excess unlabelled amino acids. The rates are plot,ted as in Fig. 1.
18 16 t ’ 14 t
: /
. : ‘\ \ .’ \ ’ \
\ \\‘, a’
,.
58-
60T4 D+ IO I5 20 25 30 Time after Infection (min)
I
315
FIG. 6. Rates of synthesis of P32 in mutants with aberrant DNA synthesis. E. coli BE at 2.0 x lOc/ml were infected at 30°C with wild type ( l ), 60amHL626 (A), or 58amEA219 ( W) at a m.o.i. of about 10. At the indicated times the infected cells were labelled with l*C-labelled amino acids for 4 min, followed by a 5.min chase with excess unlabelled amino acids. The rates are plotted as in Fig. 1.
56, 58, 60, 39, 52 and 30 mutants; data not shown for last three) exhibit, at least transiently, increased rates of gene 32 expression. The possible significance of these observations is suggested by the experiment shown in Figure 7. The increased P32 expression in 41- infection is dependent on the residual DNA synthesis in the infection because it disappears if this synthesis is blocked by introducing a second mutation in another DNA synthesis gene. Among the early genes, only the product of gene 32 has this DNA synthesis-dependent overproduction phenotype; rIIB, for example, shows no such effect (Fig. 7). Thus, the results suggest that the high rate of P32 expression in the 41- infection is caused by the abnormal DNA replication. A possible mechanism of this stimulation of expression is suggested by the nature 7
98
H.
M. KRISCH,
A. BOLLE
AND
.Lo $ lill l!!!bL
R. H.
EPSTEIN
5
4c
f 5
5/ 4-
41-
3
3-
$
“0 a, c?
2
I
.-a=. : ,J.-. --,----m--
’
:
00
5
2-
-- : 43-41-
IO 15 20 25 30 Time
after
infectton
I mini
FIG. 7. Rates of synthesis of P32 and PrIIB in gene 41, gene II-gene 43, and gene 43 mutantinfected cells. E. COGBE at 2 x lO*/ml were infected at 30°C with 41amN81 (A), 41amN81-43amB263 ( n ), or 43amB263 (0) at a m.o.i. of about 10. At the indicated times the infected cells were labelled with 14C-labelled amino acids for 4 mm, followed by a 5min chase with exoess unlabelled amino acids. The rates are plotted as in Fig. 1. The rate of synthesis of PrIIB was determined by methods identical to those used for P32.
of t,he aberrant DNA synthesis. Gene 41 mutant infection, for example, has been shown by Oishi (1968) to result in the synthesis of small fragments of single-stranded DNA. Such DNA should be capable of binding P32 with great efficiency. Similarly, the absence of the products of genes 39, 52,56, 58, or 30 results in the synthesis of limited amounts of highly discontinuous DNA (Okazaki et al., 1968 ; Kutter & Wiberg, 1968 ; Naot & Shalitin, 1972). If this DNA bound appreciable quantities of P32 to singlestranded regions the intracellular concentration of free P32 could be reduced below the threshold level required to block further synthesis of P32. A simple calculation based on the binding properties of gene 32 protein (Alberts 82;Frey, 1970) demonstrates that the production of a single phage equivalent of single-stranded DNA has the capacity to bind about 40,000 molecules of P32. This represents four times t,he total amount of P32 synthesized in a wild-type infection. It becomes clear that
Time
offer
infectmn
(min)
8. Retes of synthesis of P32 in maturation-defective and DNA synthesis-defective infections. E. coli BE at 2 x 108/ml were infected at 30°C with either X5(41amN81-42amN122-43amB2244amN82-45amElO), (0) or 55amBL292 ( n ) at a m.o.i. of 10. At the indicat,ed times the infected cells were labelled with 14C-labelled amino aoids for 4 min, followed by a 5min chase with excess unlabelled amino acids. The rates are plotted as in Fig. 1. FIG.
REGULATION
OF T4 GENE
32 PROTEIN
99
the production of even very small amounts of single-stranded DNA could account for appreciable amounts of P32 becoming tied up. If this hypothesis is correct, then it might be expected that infections with phage mutant in the maturation-defective genes would also produce increased amounts of P32. Such an infection, because of the defect in late transcription, fails to produce the late gene products which are involved in maturation and packaging of phage DNA. As a direct result, a large pool containing several hundred phage equivalents of DNA is produced. Even if only a relatively small proportion of this DNA is single-stranded, large quantities of P32 could be tied up, resulting in a stimulation of P32 synthesis. Figure 8 shows that a maturation-defective mutant (55amBL292) exhibits a marked increase in P32 synthesis late in infection (similarresults, data not shown, were obtained with a gene 33-defective infection). As would be predicted, the stimulation of P32 production by gene 55 or 33 mutants is DNA synthesis dependent ; the double mutant 42-5~5~ which cannot synthesize DNA does not overproduce P32 (data not shown). If the failure to package mat.ure DNA is in itself the entire explanation of the increased P32 synthesis in a maturation-defective infection, then it would be expected that mutations in specific late genes involved in the maturation and packaging of phage DNA would have a similar effect. Single mutants and phage with combinations of mutations in genes 20, 21, 22, 23, and 49 (essential for the packaging and maturation of DNA into completed heads) have been examined with respect to P32 synthesis. Such “head-defective” (Fig. 9) infections have a P32 synthesis pattern very similar to that of a T4D lysis-defective mut,ant. The significance of this result may be that the DNA in 55- or 33- infections has comparatively more regions of single-stranded DNA than the head-defective infections. For example, the general block in late gene expression may result in the exposure of single-stranded DNA which would normally serve as initiation sites of late transcripts, but which in the absence of late transcription are capable of binding P32. Another possibility is that although head formation is blocked in the head-defective infection, the DNA in the cell is nevertheless sequestered in some way so that it is not freely accessible for the binding of P32.
or0 5’ IO’ 15’ 20’ 25’ 30’ *35’ Tone after infection (rmn)
9. Rates of P32 synthesis in mutants defective in the packaging and maturation of DNA into completed heads. E. co&i BE at 2 x 108/ml were infected at 30°C with tamA (lysis-defective) (O), 23amB17 ( q ), 21amN90-22amE209-23amHll-IP” (+), or 21amN90.22amE209-23amHlleamH26 (A) at a m.o.i. of about 10. At the indicated times the infected cells were labelled with 14C-Iabelled amino acids for 4 min, followed by a S-min ohase with excess unlabelled amino acids. The rates are plotted &s in Fig. 1. FIG.
100
H.
M. KRISCH,
A. BOLLE
AND
R. H.
7 43-
EPSTEIN
30-
G Tmx
ofter infection
(min)
FIG. 10. Rates of P32 synthesis in the triple mutant (genes 43, 30, and 46) compared to that in the double mutant (genes 43 and 30). E. coli BE at 2 x lO*/ml were infected at 30°C with either (a) at a m.o.i. of about 10. 43amB263-3OamH39X-46amN130 (0) or 43amB263-30amH39X At the indicated times the infected cells were labelled with 14C-labelled amino acids for 4 min, followed by a B-min chase with excess unlabelled amino acids. The rates are plotted as in Fig. 1.
Further support for the competitive-binding model of the regulation of gene 32 expression is presented in the experiment shown in Figure 10. In the absence of functional DNA polymerase (gene 43) and polynucleotide ligase (gene 3U), the unreplicated parental DNA acquires numerous nicks which can be converted to gaps. Since this conversion requires the products of T4 genes 46 and 47 (putat,ive exonucleases), the triple mutant 43amB263-30amH39X-46amN130 fails to convert the nicked parental DNA into gapped DNA because it lacks gene 46 function (Riva et al., 1970b; Prashad & Hosoda, 1972). Thus, the DNA in infections with the double mutant, 43amB263-30amH39X, would be expected to contain gaps capable of binding P32 while the triple mutant DNA would only contain nicks which are incapable of binding P32 (Alberts & Frey, 1970). The data in I?igure 10 demonstrates that, as predicted, the double mutant synthesizes P32 at an increased rate while the triple mutant fails to produce P32 at elevated rates. At very late times this difference between the two infections becomes more appreciable. At these times Prashad & Hosoda (1972) have been able to demonstrate that there is significant amounts of gapping in the unreplicated parental 43--3O- DNA while the 43-30--46- DNA has much less.
8. Discussion The experiments reported here demonstrate that mutations in gene 32 result in markedly increased rates of synthesis of gene 32 protein. This unusually high rate of synthesis is not observed in cells mixedly infected with equal multiplicities of wild-type and gene 32 mutant phage. A model for the regulation of gene 32 expression which assumes that P32 is involved in the control of its own expression provides a coherent explanation for the various patterns of P32 synthesis observed. For example, P32 could act by blocking transcription of gene 32 messenger RNA. Thus,
REGULATION
OF T4 GENE
32 PROTEIN
101
P32 would be its own repressor and would bind specifically to a gene 32 operator in addition to its non-specific binding to single-stranded DNA. The extent of P32 expression would be determined by the competition of the two different binding substrates (single-stranded DNA and gene 32 operator DNA) for available P32. Another possible mechanism for the control of P32 synthesis would operate at the level of translation. By analogy with the regulation of RNA synthetase by RNA phage coat proteins (Eggen & Nathans, 1969; Bernardi & Spahr, 1972) P32 might bind to gene 32 mRNA and hinder its translation. Again the level of gene 32 expression would depend upon the competition for P32 between the two P32-binding substrates, in this case single-stranded DNA and a specific site on the gene 32 mRNA. Although our experiments cannot exclude the possibility of the direct involvement of other gene products in the regulation of gene 32 expression, the hypothesis of direct self-regulation by P32 is attractive for a number of reasons. Since both (P43 and P32) of the autoregulatory proteins found in T4 bind to DNA, it is possible that only minimal structural modifications were necessary to give them the additional specificity of binding to permit function as a repressor. As far as is known, the role of P32 in recombination, repair, and replication involves only the stabilization of single-stranded DNA regions. It is therefore tempting to imagine that P32 synthesis is regulated by the competitive binding reactions discussed above. Indeed, it is difficult to imagine a simpler way of regulating P32 synthesis to ensure that its level is sufficient for the amount of single-stranded DNA present in the infected cell. One aspect of the pattern of P32 synthesis is unexpected: although the rate of P32 synthesis increases markedly in infections where functional P32 is absent, even 30 to 40 minutes after infection the rate is still increasing. In comparison, the full expression of the lac or tryp operons of E. coli is obtained within minutes after derepression (Monod et al., 1952; Morse et al., 1968). It is possible that the pattern of P32 synthesis under what we suppose to be “de-repressed” conditions is due to competition between gene 32 mRNA and other mRNA species for available ribosomes. Another possibility is that a rather long time is required for an alteration of the translational or transcriptional machinery necessary for the full expression of gene 32. The findings reported here on the regulation of P32 synthesis have considerable bearing on the previously published work of Sinha & Snustad (1971) and Little (1973). In suggesting that P32 has a “stoichiometric” role in DNA synthesis, Sinha $ Snustad cite experiments in which the proportion of functional gene 32 copies was varied by infecting a non-permissive host with different ratios of gene 32 amber and wild-type phage. In their interpretation of these experiments it was assumed that the extent of synthesis of functional P32 would be directly related to the proportion of wild-type alleles in a given mixed infection. Self-regulation invalidates this assumption and makes the interpretation of these results more complicated. Nevertheless, the effects they observed on DNA synthesis are not at variance with what might be expected on the basis of our experiments. We interpret their data to suggest that P32 regulation allows compensatory P32 synthesis in an infection initially deficient in P32. We would expect that such compensation, however, would not occur immediately and would be highly efficient only in a limited range. Thus, even if P32 function was eventually provided, the substantial delay in its appearance
102
H.
M. KRISCH,
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AND
R. H.
EPSTEIN
might permit some irreversible damage to occur to the DNA. Indeed, the experiments of Little (1973) indicate that with temperature-sensitive mutants in gene 32 the absence of functional P32 early in infection results in the loss of the ability of the infected cell to make progeny. The recent work on pseudorevertants of gene 32 mutants is also in accord with our results. Little (1973) took advantage of the observation that gene 32 amber mutants do not form plaques on ochre suppressing hosts, which suppress amber termination codons at about the 50/, level (Brenner t Beckwith, 1965). Little selected second-site mutants which now permit plaque formation of gene 32 amber mutants on these hosts. The DNA synthesis after infection with the double mutants shows an appreciable lag and then proceeds at the wild-type rate. The second-site mutants (called “SUD”), by themselves, have a phenotype similar to the DNA-Delay mutants (Yegian et al., 1971). A hypothesis explaining Little’s results in terms of the present findings is that the “SUD ” mutation prevents irreversible DNA damage and delays the onset of rapid DNA synthesis sufficiently so that when it finally starts a normal or near normal complement of P32 has accumulated due to P32 self-regulation. Thus, late in infection replication can proceed at a normal rate. An interesting question raised by the work reported here is why P32 should exhibit the special regulatory properties described. The processes of replication, recombination and repair all generate single-stranded regions of DNA to which P32 can bind and the relative importance of these processes is likely to vary significantly in the different hosts and environments that T4 encounters in nature. Since P32 is normally required in very large amounts, a mechanism to ensure an adequate supply of this gene product may be essential. For a number of biochemical reactions related to DNA synthesis the effects of insufficient levels of required enzymes might be overcome without major increases in enzyme concentration if the period during which the enzyme acts is extended. In contrast, because of the way it functions, it seems unlikely that the activity of P32 could be increased without increasing its concentration. Therefore, the ability to increase specifically the amount of P32 available to function in repair or recombination without serious impairment of DNA replication capacity could be advantageous. We have not yet explored possible examples of natural situations where we would expect gene 32 self-regulation to be evident (small doses of U.V. radiation, different hosts, etc.). However, the hypothesis concerning P32 regulation suggests explanations for some phenotypes of mutants whose effects may be analogous to naturally occurring situations. Thus, the DNADelay phenotype in 58- and ligase--rIIinfections may be an indirect consequence of the high rate of recombination in such infections (Berger et al., 1969 ; Krisch et al., 1971). Early in these infections P32 may be limiting for replication and only later, when an adequate supply of P32 has been accumulated, might both normal replication rates and much increased (four- to fivefold) rates of recombination be accommodated. As has been implied above, the self-regulation of P32 may play an important role in optimizing the rate of DNA replication in the particular growth conditions represented by a given host or environment. This suggestion leaves open the question of how the initiation of replication is regulated, although the level of P32 presumably will affect initiation of replication as well as chain elongation. Recently, Sompayrac & Maalse (1973) have proposed, on theoretical grounds, that the initiation of bacterial chromosome replication is regulated by a protein whose intracellular
REGULATION
OF T4 GENE
32 PROTEIN
103
concentration is held constant, independent of growth rate, by means of an autorepressible operon. Should the DNA-unwinding protein or a DNA polymerase of E. coli be self-regulatory as they are in phage T4, then either of these proteins would be an ideal candidate for such an initiator. The results presented in this paper along with those of others (Russel, 1973 ; Smith & Magasanik, 1971; Sirotnak, 1971; Yoshida, 1970; Goldberger, 1974 (review)) suggest that self-regulation may be a considerably more common phenomenon than previously thought. In many cases, self-regulation may be the preferred alternative to more complex, multi-component control systems. Thus, it may be especially interesting to examine the regulation of such proteins of E. coli as RNA polymerase, the ribosomal proteins and regulatory gene products. This research was supported by a grant from the Fonds National Suisse de la Recherche Scientificlue (no. 3.836.72). One of us (H. M. K.) is a postdoctoral fellow of the National Institutes of Health (U.S.A.), Institute of General Medical Sciences (FO 2 GM52898). The authors are extremely grateful to A. Coppo, J. Pulitzer and A. Rifat for sharing their early observations on P32 synthesis. B. Alberts, T. Mattson, M. Russel, 0. Seizer and N. Hamlett contributed numerous valuable discussions. We also thank D. Berg, B. Bird, D. Kolakofsky, G. Seizer and G. Smith for their many helpful suggestions concerning the manuscript. REFERENCES Alberts, B. M. & Frey, L. (1970). Nutuure (London), 227, 1313-1318. Berger, H., Warren, A. J. & Fry, K. E. (1969). J. Viral. 3, 171-175. Bernardi, A. & Spahr, P. F. (1972). Proc. Nat. Acad. Sk., U.S.A. 69, 3033-3037. Brenner, S. & Beckwith, J. R. (1965). J. Mol. Biol. 13, 629-637. Champe, S. P. & Benzer, S. (1962). Proc. Nat. Acad. Sci., U.S.A. 48, 533-546. Delius, H., Mantell, M. & Alberts, B. M. (1972). J. Mol. BioZ. 67, 341-350. Doermann, A. H. & Boehner, L. (1970). Genetics, 66, 417-428. Eggen, K. & Nathans, D. (1969). J. Mol. BioZ. 39, 293-305. Epstein, R. H., Bolle, A., Steinberg, C. M., Kellenberger, E., Boy de la Tour, E., Chevalley, R., Edgar, R. S., Sussman, M., Denhardt, G. H. & Lielausis, A. (1963). Cold Spring Harbor Symp. Quant. BioZ. 28, 375-392. Goldberger, R. F. (1974). Science, 183, 810-816. Hosoda, J. & Levinthal, C. (1968). ‘Virology, 34, 709-727. Josslin, R. (1970). F+roZogy, 40, 719-726. Josslin, R. (1971). virology, 44, 101-107. Kozinski, A. & Felgenhauer, Z. Z. (1967). J. ViroZ. 1, 119331202. Krisch, H. M., Shah, D. B. & Berger, H. (1971). J. ViiroZ. 7, 491-498. Krisch, H. M., Hamlett, N. V. & Berger, H. (1972). Genetics, 72, 187-203. Kutter, E. M. & Wiberg, J. S. (1968). J. Mol. BioZ. 38, 395-411. Little, J. W. (1973). Virology, 53, 47-59. Monod, J., Pappenheimer, A. M. & Cohen-Bazire, G. (1952). Biochim. Biophys. Acta, 9, 648-660. Morse, D. E., Baker, R. F. & Yanofsky, C. (1968). Proc. Nat. Acad. Sci., U.S.A. 60, 14281435. Naot, Y. & Shalitin, C. (1972). J. ViroZ. 10, 858-862. O’Farrell, P. Z., Gold, L. M. & Huang, W. M. (1973). J. BioZ. Chem. 248, 549995501. Oishi, M. (1968). Proc. Nat. Acad. Sk., U.S.A. 60, 1000-1006. Okazaki, R., Okazaki, T., Sakabe, K., Sugimoto, K., Kainuma, R., Sugino, A. & Iwatsuki, N. (1968). Cold Spring Harbor Symp. Quant. BioZ. 33, 129-142. Prashad, N. & Hosoda, J. (1972). J. Mol. BioZ. 70, 617-635. Riva, S., Cascino, A. & Geiduschek E. P. (1970a). J. Mol. BioZ. 54, 85-102. Riva, S., Cascino, A. & Geiduschek, E. P. (1970b). J. MoZ. BioZ. 54, 103-119. Russel, M. (1973). J. Mol. BioZ. 79, 83-94.
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Showe, M. K. & Black, L. W. (1973). Nature New Biol. 242, 70-75. Sinha, N. K. & Snustad, D. P. (1971). J. Mol. Biol. 62, 267-271. Sirotnak, F. M. (1971). J. Bacterial. 106, 318-324. Smith, G. R. & Magasanik, B. (1971). Proc. Nat. Acad. Sci., U.S.A. 68, 1493-1497. Sompayrao, L. & Maalee, 0. (1973). Nature New Biol. 241, 133-135. Steinberg, C. M. & Edgar, R. S. (1962). Genetics, 47, 187-208. Warner, H. R. & Hobbs, M. D. (1967). ViroZogy, 33, 376-384. Wiberg, J. S., Dirksen, M. L., Epstein, R. H., Luria, S. E. & Buchanan, J. M. (1962). Proc. Nat. Acad. Sci., U.S.A. 48, 293-302. Yegian, C. D., Mueller, M., Selzer, G., Russo, V. & Stahl, F. W. (1971). VViroZogy, 46, 900919. Yoshida, A. (1970). J. Mol. Biol. 52, 483-490.
Regulation of the Synthesis of Bacteriophage T4 Gene 32 Protein H. M. KRISCH,
A. BOLI,E
AND R. H. EPSTEIN
Dipartement de Biologic MolCculaire Universitk de Gentve, Geneva, Switzerland (Received 20 February 1974) The synthesis of T4 gene 32 product (P32) has been followed by gel electrophoresis of infected cell lysates. In wild-type infections, its synthesis starts soon after infection and begins to diminish about the time late gene -expression commences. The absence of functional P32 results in a marked increase in the amount of the non-functional P32 synthesized. For example, infections of T4 mutants which contain a nonsense mutation in gene 32 produce the nonsense fragment at more than ten times the maximum rate of synthesis of the gene product observed in wild-type infections. All of the temperature-sensitive mutants in gene 32 that were tested also overproduce this product at the non-permissive temperature. This increased synthesis of the non-functional product is recessive, since mixed infections (wild-type, gene 32 nonsense mutant) fail to overproduce the nonsense fragment. Mutations in genes required for late gene expression (genes 33 and 55) as well as some genes required for normal DNA synthesis also result in increased production of P32. The overproduction in such infections is dependent on DNA synthesis; in the absence of DNA synthesis no overproduction occurs. This contrasts with the overproduction resulting from the absence of functional P32 which is not depeadent on DNA synthesis. These results are compatible with a model for the regulation of expression of gene 32 in which the synthesis of P32 is either directly or indirectly controlled by it& own function. Thus,. in the absence of ,P32 function the expression of this gene is increased as is manifest by the high rate of P32 synthesis. It is further suggested that in infections defective in late gene expression and consequently of replicated DNA, the increased P32 production is caused in the maturation by the large expansion of the DNA pool. This DNA is presumed to compete for active P32 by binding it non-specifically to single-stranded regions, thus reducing the amount of P32 free to block gene 32 expression. Similarly, the aberrant DNA synthesized following infections with mutants in genes 41,56,58,60 and 30, although quantitatively less than that produced in the maturation defective infections, can probably bind large quantities of P32 to single-stranded regions resulting in increased P32 synthesis.
The
product
DNA synthesis Amber mutants
of bacteriophage (Epstein in this
1. Introduction T4D gene 32 (P32)
is required
et aZ., 1963 ; Warner & Hobbs, gene fail
to undergo
more
than
continuously
for
T4
1967 ; Alberts & Frey, 1970). a single round of replication
in the non-permissive host (Kozinski & Felgenhauer, 1967) and a temperaturesensitive mutant ceases DNA repliciltion within a minute after shifting to the nonpermissive temperature (Riva et al., 1970a). Gene 32 appears to be unusual among 89
H.
90
M. IIRISCH,
A. BOLLE
AND
R. H.
EPSTEIN
the DNA synthesis genes in that its product is required in very large amounts (104 molecules/cell) for normal replication (Alberts & Frey, 1970). Furthermore, the level of P32 may control the amount of DNA synthesis (Sinha & Snustad, 1971). P32 binds co-operatively t)o single-stranded DNA, and facilitates the denaturation and renaturation of DNA under physiological conditions (Alberts & Frey, 1970). It has is that of stabilization of the singlebeen suggested that the role of P32 in replication stranded regions which exist transiently near the growing point of a replicating DNA molecule. Such regions presumably present an efficient template for the replication machinery (Alberts & Frey, 1970; Delius et al., 1972). The data presented in this paper suggest that gene 32 protein has a role in the regulation of its own synthesis. The absence of functional gene 32 product results in a marked increase in the amount of defective P32 synthesized. For example, in infections with T4 strains containing a nonsense mutation in gene 32, the nonsense fragments are made at ten times the maximum rate of synthesis of the gene 32 product in a wild-type infection. In addition, evidence will be presented that, because of the properties of P32, the nature and amount of DNA replication in an infected cell also have an effect on the expression of gene 32.
2. Materials and Methods (a) Bacterial strains Escherichia coli BE (sup”) from the Geneva collection was used as the host bacterium in all experiments. E. coli CR63 (sup’ 1) was used to prepare phage stocks and as the permissive host for phage containing amber mutations. (b) Bacteriophage All of the phage used in these experiments are mutants of’ T4D. The wild-type T4D and the r1 mutant, r45, used in this laboratory were originally obtained from A. H. Doermann. Except for the gene t mutant, tamA (Josslin, 1970,1971), the amber and temperature-sensitive mutants are described in Epstein et al. (1963). Phage containing combinations of these mutations were constructed by standard cross procedures (Krisch, et cd., 1972). The identification of complex genotypes was accomplished following the methods described by Doermann & Boehner (1970). The following mutant phages were used: rIr48, 3OamH39X, 32tsP7, 32tsL94, 32tsLl70, 32tsG26, 32amA453, 32amH18, 32amHL618,4ltsCB115,41amN81,42tsLB3,42aniN122,43tsP36,43amB22, 43amB263,44am.N82, 45tsL159, 45amE10, 46amN130, 55amBL292, BGamEABl, 58arnYlZA219, 60amHL626, 2lamN90, 22amE209, 23amHl1, 23amB17, eamH26, and IP” (internal protein defective, Showe & Black, 1973). (c) Media M9S
contains 0.2% Casamino acids was of host bacteria. 1962) was used as the growth medium for the broth (Steinberg & Edgar, of indicator bacteria. Phage and bacteria were plated on Hershey bot.tom
medium
(Champe
used in the preparation Hershey
preparation agar using EHA
& Benzer,
1962)
which
of stocks and for the growth
(Steinberg
& Edgar, 1962) top layer agar. (d) Experimental
Host
cells
were
grown
procedure
at 37°C to 5 X 107/ml
in M9S medium
and then
centrifuged
and resuspended at a cell density of 1.4 X 10s in M9S.l which contains only O*OZ”h Casamino acids. Unless otherwise indicated, the entire , a,t 30%. Exponential E. coli BE cells at a density added to the adsorption tube with aeration 2 min greater vol.--of M9S. 1 medium containing phage at
subsequent procedure was carried out of 1.4~ luQ/ml in M9S.l medium were before the addition of phage. A sixfold a concn of-2.3 x 10Q/ml (a multiplidity
REGULATION
OF T4 GENX
91
3.2 PROTEIN
of 10) was added to the absorption tube to begin the infection. Adsorption under these conditions was rapid and essentially all cells were infected by 3 min after the addition of phage. At the times indicated in the text and Figure legends, 5 ml of infected cells (2 x lO*/ml) were added to 0.5 ml of a mixture of 15 14C-labelled amino acids (New England Nuclear, NEC 445) at 10 ~Ci/ml. Four minutes later, 0.5 ml of 20% Casamino acids was added to terminate the labelling and after a 5-min chase, the samples were put in ice. (e) Radioactive
lysate preparation, gel electrophoresis of protein yields determination
arbd
The methods were identical to those previously used in this laboratory by Russcl (1973). The determination of relative protein yield was accomplished by exposing X-ray film (Kodak) to the dried gel slices or slab for a period of 1 to 6 days. The autoradiographs were developed and then traced with a Joyce-Loebl recording microdensitometer. The area of the peak corresponding to the protein of interest was determined by cutting out the peak and weighing it. The amount of trichloroacetic acid-precipitable counts in each lysate was determined and in general this was similar for different lysates within a single experiment. The data was normalized by dividing the peak area by the number of trichloroacetic acid-precipitable counts added to the gel. The value of the peak area/&s per min was taken as the average rate of synthesis during the period of the pulse labelling. All values shown in a given Figure (or Table) were determined using lysates from the same experiments, analysed in the same gel. The values obtained in separate experiments
have not been standardized for exposure time etc; consequently, the numerical values in different Figures are not to be compared. To compare the relative yield of the whole P32 protein and the fragment produced in 32amH18 infections, it was necessary to correct the band intensity measurements for the difference in molecular weight of the two products (P32=35,000; 32amH18= 17,000). Molecular weights were estimated from gel migration rates relative to phage T4 early proteins, the molecular weights of which had been determined (O’Farrell et al., 1973). All of the gels from which quantitative data were taken were 10% gels in which the P32 band and the adjacent PrIIBt band were well separated. A discussion of the sensitivity and sources of error in measurements of band intensities on gels is to be found infection (4 to 8 min) the label in the P32 in Russel (1973). Early in a lysis-defective band represents about 10% of the labelled protein in the gel.
3. Results Following appearance
the entry
of the phage T4 genome into a host cell, there is an ordered
of phage-specific
protein
(Hosoda
& Levinthal,
1968). Almost
immediately
after infection a group of enzymes are synthesized which are involved in the replication of phage DNA. Synthesis of these enzymes proceeds for 10 to 20 minutes at 30°C and then essentially stops (Wiberg et al., 1962). The control mechanism underlying this cessation of early enzyme synthesis is poorly understood; however, studies with mutants unable to synthesize phage DNA demonstrate that normal turn-off requires phage DNA synthesis (Wiberg et al., 1962). The sequence of gene expression in T4-infected cells can now be studied in considerable detail by the analysis of newly synthesized proteins pulse-labelled with l 4C-labelled amino acids. After a chase with unlabelled amino acids, the infected cells are lysed and a sample of the crude lysate analysed by electrophoresis on a sodium dodecyl sulphate-polyacrylamide slab gel. Autoradiographs of such gels make it possible to identify many of the T4specific proteins. Tracing of the autoradiographs with a microdensitometer permits t Abbreviations used: PrIIB, the protein product of wild-type phage T4 gene TIIB; phage mutant in such genes synthesize little if any DNA (Warner & Hobbs, 1967).
DO gene,
92
H.
M. KRISCH,
A. BOLLE
AND
R. H.
EPSTEIN
the measurement of the relative amounts of various proteins synthesised during the labelling period. The product of T4 gene 32 can be identified easily on sodium dodecyl sulphatepolyacrylamide gels. Plate I shows that lysates from infections with phage carrying any one of three different amber mutations in this gene are missing a single band which migrates with a molecular weight of 35,000. This estimate of the molecular weight of P32 is in good agreement with the value determined by Alberts & Frey (1970). In addition, each mutant lysate contains a single new band which corresponds to the nonsense fragment. The 32amH18 fragment has a molecular weight of 17,000, that of 32amHL618 is about 10,000 and the 32amA453 fragment is yet smaller. Identification of P32 on sodium dodecyl sulphate-polyacrylamide gels makes it possible to determine the time course of its synthesis by pulse-labelling at various times during infection and determining the amount of this protein synthesized during each pulse. Figure 1 shows the pattern of P32 synthesis in a lysis-defective infection. (At comparable times the patterns of P32 synthesis in wild-type and tamA (lysis-defective) infections are identical.) The synthesis of P32 commences early in infection and continues at a high rate until after the initiation of replication and of late gene expression. About 15 minutes after infection at 3O”C, P32 synthesis begins to diminish; however, unlike many other pre-replicative proteins, its synthesis is still significant long after the initiation of replication.
Time
after
mfectmn
(msn)
FIG. 1. Rates of synthesis of P32 in gene t (lysis-defective), gene 44, and gene 32 mutantinfected cells. E. c&i BE at 2 x 108/ml were infected at 3O’C with tamA ( l ), 44amN82 (m), or 32amH18 (A) at a m.o.i. of 10 and labelled at various times with 14C-labelled amino acids. Each labelling was followed by a 6-min chase with unlabelled amino acids. m.o.i., multiplicity of infection. Each point in the Figure represents the average rate of synthesis of P32 during a 4-min pulselabelling. Rates are plotted at the midpoint of each pulse interval. See Materials and Methods for additional experimental details.
When DNA synthesis is stringently blocked by a mutation in gene 44 (Warner & Hobbs, 1967), the shut-off of many early products, including P32 does not occur. Such an infection (Fig. 1) results in an extended period of P32 synthesis at nearly the maximum rate observed in a wild-type infection. A number of T4 mutants completely blocked in DNA synthesis (mutants in the DO genes: 42, 43, 44, 45) result in a similar pattern of P32 synthesis with the notable
amXS
amHI
amHl.618
ICE Y --------u-----r-----------------------
h cd Y
1
REGULATION
OF T4 GENE
32 PROTEIN
93
exception of mutants in gene 32 itself. In infections with gene 32 mutants, the absence of functional P32 results in a marked increase in the amount of the nonfunctional product synthesized. As can be seen from Figure 1, the synthesis of the defective product (nonsense fragment) occurs at a continually increasing rate throughout the course of infection. At 38 minutes after infection at 30°C with a gene 32 nonsense mutant (32amH18) the rate of synthesis of the defective product is 40 times that observed at a comparable time in the lysis-defective infection. The increased rate of P32 synthesis relative to wild-type rate is hereafter called P32 overproduction. In order to examine in more detail the synthesis of P32 in the absence of its function a series of pulse-labellings were carried out using T4 strains containing nonsense mutations in gene 32 or gene 43 (a DO gene) as well as with a strain which contains mutations in both genes. As was demonstrated in Figure 1 with a mutant in a different DNA synthesis gene (gene 44), the failure to replicate DNA results in an extended period of P32 synthesis. The data in Figure 2 demonstrates that the rate
'0
t 5 Time
1 I I I I IO 15 20 25 30 after
infection
(min)
FIG. 2. Rates of synthesis of P32 in gene 43, gene 43-gene 32, and gene 32 mutant-infected cells. E. COG BE at 2x lO’/ml were infected at 30°C with 43amB22 (a), 43amB22-32amH18 (m), of 32amH18 ( A) at a m.o.i. of about 10. At the indicated times, the infected cells were labelled with l*C-labelled amino acids for 4 min, followed by a 5-min chase with unlabelled amino acids. Rates are plotted as described in the legend to Fig. 1.
of synthesis of non-functional P32 is approximately ten times that observed in a comparable infection in which DNA synthesis is blocked by a mutation in gene 43 (43amB22). The possibility that the small amount of DNA synthesized in the 32amH18 infection (Kozinski & Felgenhauer, 1967) contributes to the overproduction of P32 has been excluded by constructing the double mutant 43amB22-32amH18 and testing it for P32 overproduction (Pig. 2). The results of this experiment demonstrate that P32 overproduction by a gene 32 mutant is not affected by a complete block in DNA synthesis.
94
H.
M. KRISCH,
A. BOLLE
AND
R. H.
EPSTEIN
TABLE 1 Gene 32 product yield relative to wild type after infection with temperature-sennsitive gene 32 mutants
t See Materials
Mutation
Relative yield? 15 to 19 min after infection
32tsp7 32tJsL94 32kL170 32tsG26
6.3 6.2 6.4 3.9
and Methods
for additional
experimental
at 42°C
details.
The data in Table 1 indicate that four temperature-sensitive mutations in gene 32 also result in increased synthesis of P32 at the non-permissive temperature. This increased P32 synthesis cannot be attributed merely to the block in DNA replication in such infections, since additional experiments indicate that the rate of P32 synthesis (in a 15 to 19 min pulse at 42°C) is at least five times greater in a 32tsP7 infection than in a 43tsP36 infection. The mutant 32tsG26, which synthesizes less P32 than the other temperature-sensitive mutants, is the leakiest of the gene 32 mutants in that, unlike the others, it makes minute plaques (on CR63) with a high efficiency at the non-permissive temperature.
4. Self-regulation The results in the previous sections indicate that P32 is either directly or indirectly involved in the regulation of its own synthesis. It has been observed previously in this laboratory that the expression of T4 gene 43 (DNA polymerase) is also regulated by its product (Russel, 1973). In view of these results it was considered important to establish whether this type of regulation was a common feature of the gene products involved in phage DNA synthesis. Experiments have been carried out at the non-permissive temperature with temperature-sensitive mutants in some of the other DNA synthesis genes. These experiments indicate that the products of gene 4I (tsCBllti), gene 42 (tsLB3), and gene 45 (tsL159) are not overproduced in the absence of their function (data not shown). These observations suggest that self-regulation is not common to all of the DNA synthesis genes but is restricted to only some of them. It has not been rigorously excluded that there are yet more self-regulatory genes in T4 (involved either in DNA synthesis or in other aspects of the life cycle). This question is currently under more extensive investigation.
5. Overproduction The relation was examined The results of sense fragment
: Recessiveness of the Gene 32 Mutant Allele
between the amount of functional P32 and the level of P32 synthesis in a series of mixed infections with wild-type and gene 32 mutants. these experiments demonstrate that the overproduction of P32 nonis recessive to the wild-type allele for gene 32. In the experiment
REGULATION
OF T4 GENE
32 PROTEIN
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shown in Plate II(a), a mixed infection of T4D+ and 32amHl8 was carried out with equal multiplicities of each type, and the amount of P32 and the 32amH18 fragment synthesized during a pulse was determined. As can be seen from Plate II(a), the presence of functional P32 greatly reduces the rate of synthesis of the amber fragment. The interpretation of this experiment is complicated by the fact that in both the wild-type control and in the mixed infection, DNA synthesis and late gene expression occur. In contrast in the other control experiment, a 32amH18 infection, neither DNA synthesis nor expression of late genes takes place. Consequently a direct comparison of the three infections is difficult. Since this complication can be circumvented by using a nonsense mutant in gene 43 instead of wild type as a control, the mixed infection was carried out with the 43amB22 strain and a strain which has mutations in both gene 43 and 32 (43amB22-32amH18). These infections (Plate II(b)) confirm the previous conclusion that functional P32 greatly reduces the rate of synthesis of the non-functional P32 fragment. In the experiment shown in Figure 3, cells were infected at the same time with different ratios of amber and wild-type phage, and the amount of P32 was measured. It is interesting to note that in the mixed infection the synthesis of P32 is the same as in the infeotion with wild type alone. In addition it is clear from the quantitative analysis of these experiments (as well as the experiments shown in Plate II) that the nonsense fragment and the intact P32 are produced in direct proportion to the input ratio.
FIG. 3. Rates of synthesis of wild-type P32 in mixed infections with wild-type and gene 32 amber mutant. E. coZi BE at 2.0 x lOs/ml were infected at 30°C with wild type alone (a), wild type and 32amH18 at an input ratio of 2 to 1 ( n ), or wild type and 32amH18 at an input ratio of 1 to 2 (A). Total multiplicity of infection in all cases was 15. At the indicated times, the infected cells were labelled with 14C-labelled amino acids for 4 min, followed by a 5-min chase with unlabelled amino acids. Rates are plott,ed as described in the legend to Fig. 1.
6. Continual Requirement of Gene 32 product for the Regulation of its Synthesis Since there is a continuous requirement of P32 for DNA synthesis, it may be asked whether the level of functional P32 regulates its own syntheGs throughout the infectious cycle. The experiment shown in Figure 4 involves an infection of E. coli BE at 30°C with a temperature-sensitive mutation in gene 32 (32tsP7), and a parallel control with a temperature-sensitive gene 43 mutant (43tsP36). After the synthesis of P32 began to diminish at 30°C the infected cells were shifted to 42~5°C. Both of the temperature-sensitive-mutant products are rapidly inactivated at the
96
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4( Time ofter infection (mini FIG. 4. Rates of synthesis of P32 before and after a shift from 30°C to 42.5’C at 22 min after infection. E. coli BE at 2 x lOs/ml were infected with 43tsP36 or 32tsP7 at a m.o.i. of 10 at 30°C. At the indicated times, portions of the culture were pulse-labelled for 4 min and then chase’d for 5 min with unlabelled amino acids. At 22 min after infection a portion of the culture was diluted with an equal volume of medium at 54’C and placed at 42.5”C. Following the temperature shift two pulse-labellings were carried out on the portion of the culture at 42.5’C. Rates are plotted as described in the legend to Fig. 1; (0) 43tsP36; (a) 32tsP7. The arrow ( 4 ) indicates the time of shift; the broken line indicates that portion of the culture shifted to 42.5% while the solid line was that portion maintained at 30°C throughout the experiment.
non-permissive temperature since DNA replication ceases within a few minutes (Riva et al., 1970a). The data in Figure 4 demonstrate that the temperature shift which renders the thermosensitive P32 inactive for replication also renders it nonfunctional for regulation of its own synthesis. Soon after the temperature shift P32 synthesis increases nearly fivefold. In the same experiment carried out with the temperature-sensitive gene 43 mutant, the increase in P32 synthesis is only about twofold. Since the temperature shift was carried out at 22 minutes, these results also indicate that the infected cell maintains the capacity to augment the synthesis of P32 in response to a deficit of the functional product well into the late period. It should be noted that even at the permissive temperature 32tsP7 synthesizes nearly twice the normal complement of P32. A possible explanation for this is thaf the 32tsP7 protein is only partially functional at the permissive temperature and twice the normal amount is required to attain the wild-type level of P32 function.
7. Role of DNA Synthesis in Gene 32 Regulation The amount of functional P32 appears to determine both the level of DNA synthesis and also the rates of its own synthesis. The nature of the relationship between DNA synthesis and the expression of gene 32 have beed investigated in a number of experiments. As was demonstrated in the experiments shown in Figures 1 and 2 infections with mutants in genes 43 or 44 (with less than 1% of the wild-type level of DNA synthesis (Warner t Hobbs, 1967)) result in an extended period of P32 gene expression. An identical pattern of P32 synthesis is obtained when DNA synthesis is gtringently blocked by use of a phage mutant. in genes 41 to 45 (see Fig. S).. As is shown in Figures 5 and 6 a number of mutants which are not rigorously blocked in DNA synthesis (Warner t Hobbs, 1967) have a quite different pattern of P32 synthesis. Mutants which synthesize a significant amount of DNA (gene 41,
REGULATION
OF T4 GENE
33
A’
li,
.-* .-*
32 PROTEIN
97
56-“A
,i
IO iime
15 20 25 30 after infection (mini (min)
FIG. 6. Rates of synthesis of P32 in mutants with aberrant DNA synthesis. E. coli BE at 2.0~ lOs/ml were infected at 30°C with T4Dr48 (a), 41amN81 ( n ), or 56amEA51 (A) at a m.o.i. of about 10. At the indicated times the infected cells were labelled with 14C-labelled Ltmino acids for 4 min, followed by a 6-min chase with excess unlabelled amino acids. The rates are plot,ted as in Fig. 1.
18 16 t ’ 14 t
: /
. : ‘\ \ .’ \ ’ \
\ \\‘, a’
,.
58-
60T4 D+ IO I5 20 25 30 Time after Infection (min)
I
315
FIG. 6. Rates of synthesis of P32 in mutants with aberrant DNA synthesis. E. coli BE at 2.0 x lOc/ml were infected at 30°C with wild type ( l ), 60amHL626 (A), or 58amEA219 ( W) at a m.o.i. of about 10. At the indicated times the infected cells were labelled with l*C-labelled amino acids for 4 min, followed by a 5.min chase with excess unlabelled amino acids. The rates are plotted as in Fig. 1.
56, 58, 60, 39, 52 and 30 mutants; data not shown for last three) exhibit, at least transiently, increased rates of gene 32 expression. The possible significance of these observations is suggested by the experiment shown in Figure 7. The increased P32 expression in 41- infection is dependent on the residual DNA synthesis in the infection because it disappears if this synthesis is blocked by introducing a second mutation in another DNA synthesis gene. Among the early genes, only the product of gene 32 has this DNA synthesis-dependent overproduction phenotype; rIIB, for example, shows no such effect (Fig. 7). Thus, the results suggest that the high rate of P32 expression in the 41- infection is caused by the abnormal DNA replication. A possible mechanism of this stimulation of expression is suggested by the nature 7
98
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5
4c
f 5
5/ 4-
41-
3
3-
$
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I
.-a=. : ,J.-. --,----m--
’
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00
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IO 15 20 25 30 Time
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I mini
FIG. 7. Rates of synthesis of P32 and PrIIB in gene 41, gene II-gene 43, and gene 43 mutantinfected cells. E. COGBE at 2 x lO*/ml were infected at 30°C with 41amN81 (A), 41amN81-43amB263 ( n ), or 43amB263 (0) at a m.o.i. of about 10. At the indicated times the infected cells were labelled with 14C-labelled amino acids for 4 mm, followed by a 5min chase with exoess unlabelled amino acids. The rates are plotted as in Fig. 1. The rate of synthesis of PrIIB was determined by methods identical to those used for P32.
of t,he aberrant DNA synthesis. Gene 41 mutant infection, for example, has been shown by Oishi (1968) to result in the synthesis of small fragments of single-stranded DNA. Such DNA should be capable of binding P32 with great efficiency. Similarly, the absence of the products of genes 39, 52,56, 58, or 30 results in the synthesis of limited amounts of highly discontinuous DNA (Okazaki et al., 1968 ; Kutter & Wiberg, 1968 ; Naot & Shalitin, 1972). If this DNA bound appreciable quantities of P32 to singlestranded regions the intracellular concentration of free P32 could be reduced below the threshold level required to block further synthesis of P32. A simple calculation based on the binding properties of gene 32 protein (Alberts 82;Frey, 1970) demonstrates that the production of a single phage equivalent of single-stranded DNA has the capacity to bind about 40,000 molecules of P32. This represents four times t,he total amount of P32 synthesized in a wild-type infection. It becomes clear that
Time
offer
infectmn
(min)
8. Retes of synthesis of P32 in maturation-defective and DNA synthesis-defective infections. E. coli BE at 2 x 108/ml were infected at 30°C with either X5(41amN81-42amN122-43amB2244amN82-45amElO), (0) or 55amBL292 ( n ) at a m.o.i. of 10. At the indicat,ed times the infected cells were labelled with 14C-labelled amino aoids for 4 min, followed by a 5min chase with excess unlabelled amino acids. The rates are plotted as in Fig. 1. FIG.
REGULATION
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32 PROTEIN
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the production of even very small amounts of single-stranded DNA could account for appreciable amounts of P32 becoming tied up. If this hypothesis is correct, then it might be expected that infections with phage mutant in the maturation-defective genes would also produce increased amounts of P32. Such an infection, because of the defect in late transcription, fails to produce the late gene products which are involved in maturation and packaging of phage DNA. As a direct result, a large pool containing several hundred phage equivalents of DNA is produced. Even if only a relatively small proportion of this DNA is single-stranded, large quantities of P32 could be tied up, resulting in a stimulation of P32 synthesis. Figure 8 shows that a maturation-defective mutant (55amBL292) exhibits a marked increase in P32 synthesis late in infection (similarresults, data not shown, were obtained with a gene 33-defective infection). As would be predicted, the stimulation of P32 production by gene 55 or 33 mutants is DNA synthesis dependent ; the double mutant 42-5~5~ which cannot synthesize DNA does not overproduce P32 (data not shown). If the failure to package mat.ure DNA is in itself the entire explanation of the increased P32 synthesis in a maturation-defective infection, then it would be expected that mutations in specific late genes involved in the maturation and packaging of phage DNA would have a similar effect. Single mutants and phage with combinations of mutations in genes 20, 21, 22, 23, and 49 (essential for the packaging and maturation of DNA into completed heads) have been examined with respect to P32 synthesis. Such “head-defective” (Fig. 9) infections have a P32 synthesis pattern very similar to that of a T4D lysis-defective mut,ant. The significance of this result may be that the DNA in 55- or 33- infections has comparatively more regions of single-stranded DNA than the head-defective infections. For example, the general block in late gene expression may result in the exposure of single-stranded DNA which would normally serve as initiation sites of late transcripts, but which in the absence of late transcription are capable of binding P32. Another possibility is that although head formation is blocked in the head-defective infection, the DNA in the cell is nevertheless sequestered in some way so that it is not freely accessible for the binding of P32.
or0 5’ IO’ 15’ 20’ 25’ 30’ *35’ Tone after infection (rmn)
9. Rates of P32 synthesis in mutants defective in the packaging and maturation of DNA into completed heads. E. co&i BE at 2 x 108/ml were infected at 30°C with tamA (lysis-defective) (O), 23amB17 ( q ), 21amN90-22amE209-23amHll-IP” (+), or 21amN90.22amE209-23amHlleamH26 (A) at a m.o.i. of about 10. At the indicated times the infected cells were labelled with 14C-Iabelled amino acids for 4 min, followed by a S-min ohase with excess unlabelled amino acids. The rates are plotted &s in Fig. 1. FIG.
100
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7 43-
EPSTEIN
30-
G Tmx
ofter infection
(min)
FIG. 10. Rates of P32 synthesis in the triple mutant (genes 43, 30, and 46) compared to that in the double mutant (genes 43 and 30). E. coli BE at 2 x lO*/ml were infected at 30°C with either (a) at a m.o.i. of about 10. 43amB263-3OamH39X-46amN130 (0) or 43amB263-30amH39X At the indicated times the infected cells were labelled with 14C-labelled amino acids for 4 min, followed by a B-min chase with excess unlabelled amino acids. The rates are plotted as in Fig. 1.
Further support for the competitive-binding model of the regulation of gene 32 expression is presented in the experiment shown in Figure 10. In the absence of functional DNA polymerase (gene 43) and polynucleotide ligase (gene 3U), the unreplicated parental DNA acquires numerous nicks which can be converted to gaps. Since this conversion requires the products of T4 genes 46 and 47 (putat,ive exonucleases), the triple mutant 43amB263-30amH39X-46amN130 fails to convert the nicked parental DNA into gapped DNA because it lacks gene 46 function (Riva et al., 1970b; Prashad & Hosoda, 1972). Thus, the DNA in infections with the double mutant, 43amB263-30amH39X, would be expected to contain gaps capable of binding P32 while the triple mutant DNA would only contain nicks which are incapable of binding P32 (Alberts & Frey, 1970). The data in I?igure 10 demonstrates that, as predicted, the double mutant synthesizes P32 at an increased rate while the triple mutant fails to produce P32 at elevated rates. At very late times this difference between the two infections becomes more appreciable. At these times Prashad & Hosoda (1972) have been able to demonstrate that there is significant amounts of gapping in the unreplicated parental 43--3O- DNA while the 43-30--46- DNA has much less.
8. Discussion The experiments reported here demonstrate that mutations in gene 32 result in markedly increased rates of synthesis of gene 32 protein. This unusually high rate of synthesis is not observed in cells mixedly infected with equal multiplicities of wild-type and gene 32 mutant phage. A model for the regulation of gene 32 expression which assumes that P32 is involved in the control of its own expression provides a coherent explanation for the various patterns of P32 synthesis observed. For example, P32 could act by blocking transcription of gene 32 messenger RNA. Thus,
REGULATION
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32 PROTEIN
101
P32 would be its own repressor and would bind specifically to a gene 32 operator in addition to its non-specific binding to single-stranded DNA. The extent of P32 expression would be determined by the competition of the two different binding substrates (single-stranded DNA and gene 32 operator DNA) for available P32. Another possible mechanism for the control of P32 synthesis would operate at the level of translation. By analogy with the regulation of RNA synthetase by RNA phage coat proteins (Eggen & Nathans, 1969; Bernardi & Spahr, 1972) P32 might bind to gene 32 mRNA and hinder its translation. Again the level of gene 32 expression would depend upon the competition for P32 between the two P32-binding substrates, in this case single-stranded DNA and a specific site on the gene 32 mRNA. Although our experiments cannot exclude the possibility of the direct involvement of other gene products in the regulation of gene 32 expression, the hypothesis of direct self-regulation by P32 is attractive for a number of reasons. Since both (P43 and P32) of the autoregulatory proteins found in T4 bind to DNA, it is possible that only minimal structural modifications were necessary to give them the additional specificity of binding to permit function as a repressor. As far as is known, the role of P32 in recombination, repair, and replication involves only the stabilization of single-stranded DNA regions. It is therefore tempting to imagine that P32 synthesis is regulated by the competitive binding reactions discussed above. Indeed, it is difficult to imagine a simpler way of regulating P32 synthesis to ensure that its level is sufficient for the amount of single-stranded DNA present in the infected cell. One aspect of the pattern of P32 synthesis is unexpected: although the rate of P32 synthesis increases markedly in infections where functional P32 is absent, even 30 to 40 minutes after infection the rate is still increasing. In comparison, the full expression of the lac or tryp operons of E. coli is obtained within minutes after derepression (Monod et al., 1952; Morse et al., 1968). It is possible that the pattern of P32 synthesis under what we suppose to be “de-repressed” conditions is due to competition between gene 32 mRNA and other mRNA species for available ribosomes. Another possibility is that a rather long time is required for an alteration of the translational or transcriptional machinery necessary for the full expression of gene 32. The findings reported here on the regulation of P32 synthesis have considerable bearing on the previously published work of Sinha & Snustad (1971) and Little (1973). In suggesting that P32 has a “stoichiometric” role in DNA synthesis, Sinha $ Snustad cite experiments in which the proportion of functional gene 32 copies was varied by infecting a non-permissive host with different ratios of gene 32 amber and wild-type phage. In their interpretation of these experiments it was assumed that the extent of synthesis of functional P32 would be directly related to the proportion of wild-type alleles in a given mixed infection. Self-regulation invalidates this assumption and makes the interpretation of these results more complicated. Nevertheless, the effects they observed on DNA synthesis are not at variance with what might be expected on the basis of our experiments. We interpret their data to suggest that P32 regulation allows compensatory P32 synthesis in an infection initially deficient in P32. We would expect that such compensation, however, would not occur immediately and would be highly efficient only in a limited range. Thus, even if P32 function was eventually provided, the substantial delay in its appearance
102
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might permit some irreversible damage to occur to the DNA. Indeed, the experiments of Little (1973) indicate that with temperature-sensitive mutants in gene 32 the absence of functional P32 early in infection results in the loss of the ability of the infected cell to make progeny. The recent work on pseudorevertants of gene 32 mutants is also in accord with our results. Little (1973) took advantage of the observation that gene 32 amber mutants do not form plaques on ochre suppressing hosts, which suppress amber termination codons at about the 50/, level (Brenner t Beckwith, 1965). Little selected second-site mutants which now permit plaque formation of gene 32 amber mutants on these hosts. The DNA synthesis after infection with the double mutants shows an appreciable lag and then proceeds at the wild-type rate. The second-site mutants (called “SUD”), by themselves, have a phenotype similar to the DNA-Delay mutants (Yegian et al., 1971). A hypothesis explaining Little’s results in terms of the present findings is that the “SUD ” mutation prevents irreversible DNA damage and delays the onset of rapid DNA synthesis sufficiently so that when it finally starts a normal or near normal complement of P32 has accumulated due to P32 self-regulation. Thus, late in infection replication can proceed at a normal rate. An interesting question raised by the work reported here is why P32 should exhibit the special regulatory properties described. The processes of replication, recombination and repair all generate single-stranded regions of DNA to which P32 can bind and the relative importance of these processes is likely to vary significantly in the different hosts and environments that T4 encounters in nature. Since P32 is normally required in very large amounts, a mechanism to ensure an adequate supply of this gene product may be essential. For a number of biochemical reactions related to DNA synthesis the effects of insufficient levels of required enzymes might be overcome without major increases in enzyme concentration if the period during which the enzyme acts is extended. In contrast, because of the way it functions, it seems unlikely that the activity of P32 could be increased without increasing its concentration. Therefore, the ability to increase specifically the amount of P32 available to function in repair or recombination without serious impairment of DNA replication capacity could be advantageous. We have not yet explored possible examples of natural situations where we would expect gene 32 self-regulation to be evident (small doses of U.V. radiation, different hosts, etc.). However, the hypothesis concerning P32 regulation suggests explanations for some phenotypes of mutants whose effects may be analogous to naturally occurring situations. Thus, the DNADelay phenotype in 58- and ligase--rIIinfections may be an indirect consequence of the high rate of recombination in such infections (Berger et al., 1969 ; Krisch et al., 1971). Early in these infections P32 may be limiting for replication and only later, when an adequate supply of P32 has been accumulated, might both normal replication rates and much increased (four- to fivefold) rates of recombination be accommodated. As has been implied above, the self-regulation of P32 may play an important role in optimizing the rate of DNA replication in the particular growth conditions represented by a given host or environment. This suggestion leaves open the question of how the initiation of replication is regulated, although the level of P32 presumably will affect initiation of replication as well as chain elongation. Recently, Sompayrac & Maalse (1973) have proposed, on theoretical grounds, that the initiation of bacterial chromosome replication is regulated by a protein whose intracellular
REGULATION
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32 PROTEIN
103
concentration is held constant, independent of growth rate, by means of an autorepressible operon. Should the DNA-unwinding protein or a DNA polymerase of E. coli be self-regulatory as they are in phage T4, then either of these proteins would be an ideal candidate for such an initiator. The results presented in this paper along with those of others (Russel, 1973 ; Smith & Magasanik, 1971; Sirotnak, 1971; Yoshida, 1970; Goldberger, 1974 (review)) suggest that self-regulation may be a considerably more common phenomenon than previously thought. In many cases, self-regulation may be the preferred alternative to more complex, multi-component control systems. Thus, it may be especially interesting to examine the regulation of such proteins of E. coli as RNA polymerase, the ribosomal proteins and regulatory gene products. This research was supported by a grant from the Fonds National Suisse de la Recherche Scientificlue (no. 3.836.72). One of us (H. M. K.) is a postdoctoral fellow of the National Institutes of Health (U.S.A.), Institute of General Medical Sciences (FO 2 GM52898). The authors are extremely grateful to A. Coppo, J. Pulitzer and A. Rifat for sharing their early observations on P32 synthesis. B. Alberts, T. Mattson, M. Russel, 0. Seizer and N. Hamlett contributed numerous valuable discussions. We also thank D. Berg, B. Bird, D. Kolakofsky, G. Seizer and G. Smith for their many helpful suggestions concerning the manuscript. REFERENCES Alberts, B. M. & Frey, L. (1970). Nutuure (London), 227, 1313-1318. Berger, H., Warren, A. J. & Fry, K. E. (1969). J. Viral. 3, 171-175. Bernardi, A. & Spahr, P. F. (1972). Proc. Nat. Acad. Sk., U.S.A. 69, 3033-3037. Brenner, S. & Beckwith, J. R. (1965). J. Mol. Biol. 13, 629-637. Champe, S. P. & Benzer, S. (1962). Proc. Nat. Acad. Sci., U.S.A. 48, 533-546. Delius, H., Mantell, M. & Alberts, B. M. (1972). J. Mol. BioZ. 67, 341-350. Doermann, A. H. & Boehner, L. (1970). Genetics, 66, 417-428. Eggen, K. & Nathans, D. (1969). J. Mol. BioZ. 39, 293-305. Epstein, R. H., Bolle, A., Steinberg, C. M., Kellenberger, E., Boy de la Tour, E., Chevalley, R., Edgar, R. S., Sussman, M., Denhardt, G. H. & Lielausis, A. (1963). Cold Spring Harbor Symp. Quant. BioZ. 28, 375-392. Goldberger, R. F. (1974). Science, 183, 810-816. Hosoda, J. & Levinthal, C. (1968). ‘Virology, 34, 709-727. Josslin, R. (1970). F+roZogy, 40, 719-726. Josslin, R. (1971). virology, 44, 101-107. Kozinski, A. & Felgenhauer, Z. Z. (1967). J. ViroZ. 1, 119331202. Krisch, H. M., Shah, D. B. & Berger, H. (1971). J. ViiroZ. 7, 491-498. Krisch, H. M., Hamlett, N. V. & Berger, H. (1972). Genetics, 72, 187-203. Kutter, E. M. & Wiberg, J. S. (1968). J. Mol. BioZ. 38, 395-411. Little, J. W. (1973). Virology, 53, 47-59. Monod, J., Pappenheimer, A. M. & Cohen-Bazire, G. (1952). Biochim. Biophys. Acta, 9, 648-660. Morse, D. E., Baker, R. F. & Yanofsky, C. (1968). Proc. Nat. Acad. Sci., U.S.A. 60, 14281435. Naot, Y. & Shalitin, C. (1972). J. ViroZ. 10, 858-862. O’Farrell, P. Z., Gold, L. M. & Huang, W. M. (1973). J. BioZ. Chem. 248, 549995501. Oishi, M. (1968). Proc. Nat. Acad. Sk., U.S.A. 60, 1000-1006. Okazaki, R., Okazaki, T., Sakabe, K., Sugimoto, K., Kainuma, R., Sugino, A. & Iwatsuki, N. (1968). Cold Spring Harbor Symp. Quant. BioZ. 33, 129-142. Prashad, N. & Hosoda, J. (1972). J. Mol. BioZ. 70, 617-635. Riva, S., Cascino, A. & Geiduschek E. P. (1970a). J. Mol. BioZ. 54, 85-102. Riva, S., Cascino, A. & Geiduschek, E. P. (1970b). J. MoZ. BioZ. 54, 103-119. Russel, M. (1973). J. Mol. BioZ. 79, 83-94.
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Showe, M. K. & Black, L. W. (1973). Nature New Biol. 242, 70-75. Sinha, N. K. & Snustad, D. P. (1971). J. Mol. Biol. 62, 267-271. Sirotnak, F. M. (1971). J. Bacterial. 106, 318-324. Smith, G. R. & Magasanik, B. (1971). Proc. Nat. Acad. Sci., U.S.A. 68, 1493-1497. Sompayrao, L. & Maalee, 0. (1973). Nature New Biol. 241, 133-135. Steinberg, C. M. & Edgar, R. S. (1962). Genetics, 47, 187-208. Warner, H. R. & Hobbs, M. D. (1967). ViroZogy, 33, 376-384. Wiberg, J. S., Dirksen, M. L., Epstein, R. H., Luria, S. E. & Buchanan, J. M. (1962). Proc. Nat. Acad. Sci., U.S.A. 48, 293-302. Yegian, C. D., Mueller, M., Selzer, G., Russo, V. & Stahl, F. W. (1971). VViroZogy, 46, 900919. Yoshida, A. (1970). J. Mol. Biol. 52, 483-490.