Proc. Nat. Acad. Sci. USA
Vol. 72, No. 12, pp. 4886-4890, December 1975 Biochemistry
Synthesis of specific functional messenger RNA in vitro by phage-SPO1-modified RNA polymerase of Bacillus subtilis (protein synthesis in vitro/rifampicin)
MARSHAL SWANTON*, DONNA H. SMITH, AND DAVID A. SHUB Department of Biological Sciences, State University of New York at Albany, Albany, N.Y. 12222
Communicated by Cyrus Levinthal, September 30,1975
ABSTRACT RNA polymerase (nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) was purified from rifampicin-resistant Bacillus subtifis, from both uninfected cells and cells infected with bacteriophage SPOl. The enzyme from infected cells lacked all traces of the sigma subunit, contained several polypeptides absent from the enzyme made in uninfected cells, and had an altered template specificity in a transcription assay. A cell-free protein synthesizing system from Escherichia coli, when poisoned with rifampicin, was completely dependent on addition of either of these RNA polymerase preparations for DNA-dependent protein synthesis. Under these conditions, the SPOI-modified RNA polymerase preferentially stimulated the synthesis of functional mRNA for the phage enzyme dCMP deaminase (deoxycytidylate aminohydrolase, EC 3.5.4.12), whereas unmodified B. subtilis RNA polymerase could stimulate synthesis of this mRNA in small quantity and only after prolonged incubation. This mRNA belongs to a class of phage transcripts (m) which cannot be transcribed in vivo in the absence of phage-specific protein synthesis.
Competitive RNA-DNA hybridization is a very powerful tool which can be used to determine the relative amount of specific nucleotide sequences in a population of in vitro synthesized RNA molecules. However, it is much more difficult to obtain precise information about the details of the specificity of synthesis, i.e., do the in vitro synthesized RNA molecules start and end at the correct place?; is the fidelity of transcription high?; etc. These questions can best be answered by using as the assay for selective transcription the ability of the in vitro synthesized RNA molecules to function as the message for a specific protein in a coupled transcription-translation cell-free system (6). We have previously shown (7) that such an Escherichia coli cell-free system is capable of using SPOl DNA or RNA to stimulate very efficient protein synthesis. In this report, we show that purified, phage-modified Bacillus subtilis RNA polymerase transcribes SPO1 DNA in this system preferentially into RNA which functions as message for the middle (m) phage-specific enzyme dCMP deaminase (deoxycytidylate aminohydrolase, EC 3.5.4.12). In addition we show that B. subtilis and SP01-modified RNA polymerases transcribe a qualitatively different set of functional, SP01-specific, messenger RNAs in this system.
Transcription of the genome of bacteriophage SP01 has been extensively studied by competitive RNA-DNA hybridization (1). Six classes of transcripts have been described, which are independently controlled with respect to the turning on or turning off of their synthesis. Two of these (e and em) are synthesized after infection without detectable lag, and are subsequently repressed if phage protein synthesis is allowed to occur. Three additional classes of RNA (m, mll and m21) are made independently of phage DNA replication, but require prior phage protein synthesis. In particular, the protein product of cistron 28 is required for m and m1l expression, and the protein products of cistrons 33 and 34 are required for M21 RNA synthesis (2, 3). After phage DNA synthesis begins, another population of transcripts (1) is made. The existence of three pre-replicative classes of phagespecific transcripts (m, m1l, and M21), whose synthesis is under phage genetic control, suggested the possibility that mature phage DNA extracted from purified virus might be an appropriate template for a new RNA polymerase (nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) of the correct specificity for these classes. Since it had been shown that the rifampicin sensitivity of RNA synthesis throughout SP01 infection was determined by the sensitivity of the host's RNA polymerase (4) it was logical to expect that the new specificity for middle RNA synthesis should reside in the host's RNA polymerase, which had been modified in some way. Duffy and Geiduschek (5) demonstrated that the expected in vitro selectivity for phage middle transcripts does, indeed, reside in an enzyme which contains the host core and other peptides which are made after phage infection. *
MATERIALS AND METHODS Bacteria and Phage. Bacillus subtilis 1005 (rifR), a derivative of W168 resistant to high levels of rifampicin, was obtained from Dr. T. Leighton. F21, a suppressible nonsense mutant of phage SP01, and its permissive host B. subtilis HAlOiB, were obtained from Dr. E. P. Geiduschek. Isolation of RNA Polymerase. B. subtilis 1005 was grown in 12 liter batches in a New Brunswick fermentor. The medium (2XLB) contains (weight/volume): 2.5% Bacto tryptone (Difco), 2.0% yeast extract (Difco), 0.3% NaH2PO4, and 3% glucose. This medium supports logarithmic growth (with vigorous aeration) to an OD540 of greater than 2. Cultures were harvested at OD540 of 1.5 or infected with SP01 (multiplicity of infection = 5.0) at OD540 of 1.0 and harvested after 15 min. The protease inhibitor phenylmethylsulfonyl fluoride was added at 0.5 mM to uninfected cultures 10 min before harvest and to infected cultures 5 min before harvest. All cultures were harvested by pouring over ice and collected in a refrigerated continuous flow centrifuge. Cells were washed once with buffer I [10 mM Tris-HCl (pH 8.0), 50 mM KC1, 10 mM MgCl2, 0.1 mM dithiothreitol, 0.1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10% (vol/vol) glycerol] and stored in liquid N2. We used a modification of the procedure of Duffy and Geiduschek (8) for enzyme purification. All operations were performed at 40. Cell pellets (20 g or more) were resuspended in 2 volumes of buffer I, homogenized in a Sorvall omnimixer, and passed through a French pressure cell at 10-
Present address: Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colo. 80302.
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Proc. Nat. Acad. Sci. USA 72 (1975)
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Table 1. Purification of RNA polymerase of B. subtilis Uninfected cells (20.3 g)
Crude lysate High-speed supernatant Gel filtration DEAE-cellulose DNA-cellulose
SPO1-infected cells (31.7 g)
Total units
% Recovery
288
100
803
100
300 178 84 54
104 62 29 19
908 482 242 146
113 60 30 18
Vi.--l--
Total % units Recovery
_-VII
_._
-VI -IV
RNA polymerase activity was determined after various stages of purification, described in Materials and Methods. Activity is expressed as units/g of packed cells.
-111
-1I12,000 pounds/inch2 (69-83 MPa). Debris was removed at 20,000 X g for 30 min. Solid (NH4)2SO4 was added to the supernatant ("crude lysate") to 25% saturation and a highspeed supernatant was obtained by centrifugation at 136,000 X g for 3 hr. This supernatant was brought to 70% saturation with solid (NH4)2SO4; the precipitate was collected by centrifugation at 48,000 X g for 30 min, dissolved in 15-20 ml of buffer II [10 mM Tris-HCl (pH 8.0), 1.0 M KCl, 0.1 mM dithiothreitol, 0.1 mM EDTA, 15% (vol/vol) glycerol], and applied to a Bio-Gel A-1.5m agarose column (2.5 X 74 cm) equilibrated with buffer II. The column was eluted at 30 ml/hr and 5 ml fractions were collected. Active fractions were pooled, dialyzed against buffer II (without KCl) to reduce KC1 concentration, and precipitated by adding solid (NH4)2SO4 to 70% saturation. The precipitate was collected by centrifugation, resuspended in 15-20 ml of buffer III [10 mM Tris-HCl (pH 8.0), 50 mM KCI, 0.1 mM dithiothreitol, 1 mM EDTA, 15% (vol/vol) glycerol] and applied to a DEAEcellulose (Whatman DE-52) column (2.5 X 11 cm) equilibrated with buffer III. The RNA polymerase activity was eluted with a linear 0.05-0.5 M KCI gradient at 50 ml/hr. Active fractions were pooled, diluted to less than 0.15 M KCI, and applied to a calf thymus DNA (Sigma, type I) column (2 X 11 cm) containing 350 ,ug of DNA per packed ml (9). The column was equilibrated with buffer III (containing 0.15 M KCI) and RNA polymerase activity was eluted with a linear 0.15-1.0 M KCI gradient. Active fractions were pooled, concentrated in an Amicon ultra filtration device using a UM10 filter, dialyzed against storage buffer [10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.1 mM dithiothreitol, 50% (vol/vol) glycerol] and stored at -200. Enzyme Assays. RNA polymerase was assayed in 0.1 ml reaction mixtures containing: ATP, GTP, CTP, UTP, 1 mM each; 50 mM Tris-HCl, pH 8.0; 50 mM KCI; 10 mM MgCI2; 0.5 mM dithiothreitol; [5-3H]UTP, 5 ,uCi/jimol; SP01 DNA, 50 jig/ml. Reaction mixtures were assembled on ice and started by warming to 370. Reactions were terminated after 10 min by precipitating with cold 5% CI3CCOOH, filtered, and counted in a scintillation counter. One unit is defined as incorporation of 1 nmol of UTP in 10 min. dCMP deaminase was assayed as previously described (7). Phage Nucleic Acid. Phage SP01, its mutant sus F21, and E. coli phage T4D (the gift of Drs. R. Trimble and F. Maley) were purified on a CsCI step gradient and DNA was extracted with phenol as previously described (7). RNA was prepared from B. subtilis harvested 10 min after phage infection as previously described (7). Gel Electrophoresis. Sodium dodecyl sulfate polyacrylamide slab gels were prepared (10) with 15% acrylamide
A B C
D E F
FIG. 1. Sodium dodecyl sulfate gel electrophoresis of RNA polymerase. Sodium dodecyl sulfate acrylamide gels (15%) were prepared using the buffer system of Laemmli (13). Enzyme from SP01-infected B. subtilis, A: 1.74 units; B: 0.87 units; C: 0.43 units. Enzyme from uninfected B. subtilis, D: 1.95 units; E: 0.98 units; F: 0.49 units.
and bisacrylamide/acrylamide = 0.0058 (11). Gels were run for 4-5 hr, fixed and stained in: 50% (vol/vol) methanol, 7% acetic acid, and 0.2% Coomassie brilliant blue, and destained in methanol, acetic acid, water (5:1:5). Stain intensity was determined by scanning the gels with a Gilford spectrophotometer at 540 nm. Areas under peaks were cut out and weighed. In Vitro Protein Synthesis. Conditions were as previously described (7, 12) except that, when purified B. subtilis RNA polymerase was used to stimulate protein synthesis, the enzyme and DNA were incubated with triphosphates, separately for 2 min at 370 before adding the other components of the cell-free protein synthesis system. Incubations were at 370 for an additional 30 min. Protein synthesis was followed by incorporation of [2-14C]leucine (54 gCi/Asmol) into hot CL6CCOOH-insoluble form (7). at 100-125 V
RESULTS DNA-dependent RNA polymerase was purified from phageSPOl-infected or uninfected B. subtilis as described in Materials and Methods. The two preparations behaved identically at each step of the purification. The total units and percent recovery at each step are shown in Table 1. Specific activities were 430 units/mg for the B. subtilis enzyme and 270 units/mg for the enzyme prepared from infected cells. The enzyme preparation obtained after DNA-cellulose chromatography was subjected to sodium dodecyl sulfateacrylamide gel electrophoresis (Fig. 1). The gels were scanned and the relative concentrations of the principal polypeptides were determined (Table 2). Molecular weights of these polypeptides were approximated by comparing their mobilities with a set of proteins of known molecular weight. These are only estimates, since it is known (13) that this gel system can give incorrect molecular weight values. In addition to the polypeptides of B. subtilis core RNA polymerase ( alj3',a), we observe four additional polypeptides in both the uninfected and SPOl-infected enzyme preparations. One of these (peptide VIII, molecular weight 110,000) is present in much higher amount in the SPOl-infected enzyme, and a polypeptide of this molecular weight has been reported as a
Proc. Nat. Acad. Sci. USA 72 (1975)
Biochemistry: Swanton et al.
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Table 2. Relative concentration and molecular weights of polypeptides associated with B. subtilis and SP01-modified RNA polymerase
Table 3. Template specificity of RNA polymerase Relative activity DNA
Concentration relative to a2 PP
Peptide
Uninfected
Sigma I II III IV V VI VII VIII
0.33 0.48 0.77
0.35 0.05
Uninfected
SP01-modified
1.0 0.99 0.47
1.0 0.10 0.28
SPOl
SP01infected
Molecular weight
1.11 1.37 0.45 0.15 0.37 1.02 0.93 0.45
11,000 12,500 17,000 19,500 23,500 26,000 47,000 110,000
The sodium dodecyl sulfate-acrylamide gel from Fig. 1 was scanned and areas under peaks were quantitated. Relative molar concentrations were normalized to the molar concentration of a2O3'. Molecular weights were estimated using standards: bovine serum albumin (68,000), glutamate dehydrogenase (53,000), gamma globulin heavy chain (55,000), yeast alcohol dehydrogenase (37,000), chymotrypsinogen (25,700), and myoglobin (17,200).
contaminant of B. subtilis RNA polymerase purified on DNA-cellulose columns (14). The other three peptides resemble new subunits recently found in B. subtilis RNA polymerase (8, 16). The RNA polymerase preparation from SP01-infected cells differs from the uninfected enzyme in several ways. There is no detectable sigma subunit in the SPO-infected enzyme, even though enzyme prepared by identical methods from uninfected cells yields a large amount of sigma. There are, however, four new polypeptides unique to the enzyme prepared from SPO-infected cells. One of them (peptide IV) is present in a low molar ratio. The others (peptides III, VI, and VII) are present in sufficiently high molar ratio to be likely candidates for new phage-specified enzyme subunits. New polypeptides associated with purified SP01- (or SP82-) modified RNA polymerase have been reported (8, 16, 17). Several of these have molecular weights similar to our peptides III, IV, and VI. A polypeptide similar in size to peptide VII has not previously been reported as a component of highly purified phagemodified enzyme. One can see, from Table 3, that the SP01-modified RNA
T4 Calf thymus
Uninfected RNA polymerase (19.5 units/ml) and SP01-modified RNA polymerase (17.4 units/ml) were assayed as described in Materials and Methods, using several templates at DNA excess. Activities are expressed relative to SP01 DNA.
polymerase has a dramatically different template-specificity than the B. subtilis enzyme. Whereas both phage SP01 and T4 DNAs are excellent templates for the enzyme from uninfected cells, the SP01-modified enzyme retains high activity on SP01 DNA, but has virtually lost the ability to transcribe T4 DNA. Both enzymes are active on calf thymus DNA although at reduced levels compared to SP01 DNA. Our objectives in this work were to determine: (1) if RNA synthesized by SP01-modified RNA polymerase could function in protein synthesis, (2) whether these proteins were complete and functional (enzymatically active) and, (3) whether the proteins were of the correct specificity (middle). Table 4 shows that when a cell-free DNA-dependent protein synthesizing system is poisoned with the RNA polymerase inhibitor rifampicin overall protein and enzyme (dCMP deaminase) synthesis fall to very low levels. These levels can be restored by adding RNA polymerase from uninfected B. subtilis which is resistant to rifampicin. However, a dramatic increase in dCMP deaminase synthesis, over that of the standard cell free system, is observed when the added RNA polymerase is SP01-modified. This preferential stimulation of synthesis of dCMP deaminase by SP01modified RNA polymerase was encouraging, since dCMP deaminase has been shown to be an enzyme under middle (m) mRNA control (7). The SP01 mutant sus F21 (cistron 28) is defective in transcription of middle messages (2) and does not make dCMP deaminase in nonpermissive infections (3). DNA from sus F21 is as competent as wild-type SP01 DNA in directing synthesis of dCMP deaminase mRNA by the B. subtilis or SP01-modified RNA polymerase (Table 4). This shows that the in vitro synthesized product of gene 28 is not acting in vitro in the synthesis of dCMP deaminase message by either enzyme.
Table 4. In vitro protein synthesis stimulated by uninfected or SP01-modified B. subtilis RNA polymerase
Rifampicin
Template SP01 DNA SP01 DNA SP01 DNA SP01 DNA F21 DNA F21 DNA 10 min RNA
RNA polymerase
10
jig/ml +
SP01-infected Uninfected SP01-infected Uninfected
+ + + + + +
SP01-infected
+
Uninfected
+
dCMP deaminase
(units/S50 l) 244 0 1270 228 1536 196 1846 8 46 0
Leucine incorporation (pmol/50, l) 1327 73 2050 1002 2040 948 669 101
Cell-free protein synthesis was carried out as described in Materials and Methods. Concentrations of added template were: SP01 DNA, 45 Mg/ml; F21 DNA, 35 Mg/ml; RNA extracted from SP01-infected cells at 10 min after infection, 1.2 mg/ml. RNA polymerase from uninfected B. subtilis was added at 19.5 units/ml and SP01-modified RNA polymerase was added at 17.4 units/ml. In determining dCMP deaminase activity, the enzyme blank, equivalent to 54 units/50 Ml, has been subtracted from all values.
Biochemistry: Swanton et al.
Proc. Nat. Acad. Sci. USA 72 (1975)
4889
.
X----X
c
x
500 x
x d
-6
0
-
I
10 Minutes
20
FIG. 2. Time course of synthesis of dCMP deaminase in vitro. In vitro protein synthesis reactions were programmed with SP01 DNA (45 gtg/ml). Uninhibited cell-free system, no addition (0); plus rifampicin (10 gg/ml) and B. subtilis RNA polymerase (-); plus rifampicin (10 gg/ml) and SP01-modified RNA polymerase (Xi. At various times after starting the reactions, 50 gl samples were removed into actinomycin (10 ug/ml) and incubation was continued for a total of 30 min. dCMP deaminase activity was determined and expressed as units/50 Al.
If SPOI-modified RNA polymerase initiates transcription at middle promotors of SP01 DNA, one would expect dCMP deaminase to appear very rapidly in protein synthesis incubations stimulated by this polymerase. On the other hand, if dCMP deaminase synthesis in reactions stimulated by uninfected B. subtilis RNA polymerase is the result of readthrough by this enzyme, then there should be a longer lag in its appearance. The experiment described in Fig. 2 confirms these expectations and supports our conclusions that the SP01-modified RNA polymerase we have prepared exhibits selectivity for middle phage promotors. To gain a more general view of transcription specificity of these two RNA polymerase preparations, cell-free protein synthesis was carried out with [2-14C]leucine. Reactions mixtures were separated by gel electrophoresis and radioactive proteins were detected by radioautography. Fig. 3 shows that both enzyme preparations direct the synthesis of proteins of discrete molecular weights. Several proteins appear to be synthesized in response to both RNA polymerases, but there are other proteins whose mRNA is a product of one of the enzymes, and not of the other. The SPO-modified RNA polymerase exhibits, therefore, both the positive and negative control functions required during the middle phase of SP01 development. A fuller characterization of the radioactive proteins displayed in Fig. 3 will be presented elsewhere.
DISCUSSION We have purified RNA polymerase from B. subtilis infected by phage SPOl using standard techniques. Our enzyme preparation contains all the polypeptides present in the host cell RNA polymerase, with the exception of the sigma subunit. There are, in addition, three new polypeptides which copurify with the phage enzyme. The results of Table 3 show that the phage-modified enzyme has an altered template specificity. Although the phage-modified RNA polymerase is even more active than host enzyme on SPOl DNA, it is virtually inactive when T4 DNA is the template. RNA polymerases of E. coli and B. subtilis are both' extremely active on the SP01 DNA template and both make identical populations of transcripts, whether assayed by competition hybridization (15) or by gel electrophoresis of polypeptides synthesized in the coupled transcription-translation system used in this work (in preparation). Since the SPO-phage-modified RNA polymerase retains high activity on SP01 DNA, the loss of activity on the
3 1 2 FIG. 3. Gel electrophoresis of SP01 proteins synthesized in vitro. Reactions were carried out with SP01 DNA and [2-'4C]leucine as described in Materials and Methods and the legend to Table 4. After 30 min, samples were mixed with an equal volume of 2-fold concentrated "final sample buffer" of Laemmli (13) and heated for 1 min at 900. Gel electrophoresis was carried out as described in Materials and Methods. After destaining the gels were dried (10) and exposed for radioautography with Kodak No Screen x-ray film. Columns 2 and 3 each represent duplicate reactions. (1) E. coli cell-free system plus rifampicin. (2) Plus SP01-modified RNA polymerase, 17.4 units/mL (3) Plus B. subtilis RNA polymerase, 19.5 units/ml.
T4 DNA template indicates that this enzyme has an altered promotor specificity. B. subtilis holoenzyme is equally active with SPO or T4 DNA, and the lack of activity of SP01modified enzyme on T4 DNA could be a consequence of the loss of sigma factor. In fact, it has been shown that the behavior of SP01-modified RNA polymerase on heterologous template is similar to that of core polymerase, including the ability to be stimulated by B. subtilis sigma factor (8). Our previous work (7) has shown that the phage-induced enzyme dCMP deaminase is a protein whose mRNA is middle (m) in the terminology of Gage and Geiduschek (1). That is, transcription of mRNA for dCMP deaminase is initiated only after the third minute of infection, reaches a maximum level in the cell at about the 10th minute, declines rapidly after the 13th minute, and is absolutely dependent on phage protein synthesis (7). A coupled transcriptiontranslation system system (12) from E. coli could synthesize dCMP deaminase when primed with SPOl DNA, but only after a long lag and at relatively low levels (7). Since the RNA initiations which ultimately led to dCMP deaminase synthesis occurred without lag, we interpreted the synthesis of dCMP deaminase mRNA as resulting from read-through transcription by the E. coli RNA polymerase. In the present work, we have continued to utilize the E. coli cell-free system for translation, but have poisoned the endogenous E. coli RNA polymerase with rifampicin, making DNA-dependent protein synthesis entirely dependent on addition of purified RNA polymerase extracted from rifampicin-resistant B. subtilis. Our expectation was that addition of RNA polymerase prepared from vegetative cells of B. subtilis would produce a reconstituted system identical to the uninhibited E. coil system with regard to dCMP deaminase synthesis. On the other hand, if the SPO-phage-modified RNA polymerase had retained its' promotor specificity in vitro, we expected to observe synthesis and translation of .dCMP deaminase mRNA without lag and at higher levels. The results of our experiments have confirmed our predictions. Using SP01 DNA as a template, the uninhibited E. coli cell-free system synthesizes some dCMP deaminase
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Biochemistry: Swanton et al.
after a lag of 10 min, in agreement with our previous results (7). When E. coli RNA polymerase is poisoned by rifampicin, addition of RNA polymerase from uninfected B. subtilis restores protein synthesis to a high level, but allows the synthesis of barely detectable levels of dCMP deaminase (Table 4). The amount of enzyme ultimately produced is so low that we are unable to extrapolate an initial time of appearance; the earliest time at which enzyme was detected was 9 min (Fig. 2). The amino-acid incorporation shows no lag and produces the same set of polypeptides of discrete molecular weights as does the uninhibited cell-free system (manuscript in preparation). The virtual absence of transcription of dCMP deaminase mRNA may be due to higher fidelity in termination of the B. subtilis RNA polymerase than the E. coli enzyme. However, addition of SPOI-modified RNA polymerase to the rifampicin-poisoned cell-free system stimulates protein synthesis and dCMP deaminase synthesis to high levels (Table 4), also without any appreciable lag (Fig. 2). When the radioactive proteins made in this system are analyzed by gel electrophoresis, they appear to be a completely different population from those made in response to stimulation by RNA polymerase from uninfected cells (Fig. 3). Therefore, we conclude that SPOI-modified RNA polymerase exhibits the same alteration of promotor selectivity in vitro as it does in vivo, and that much of the subsequent transcription is of messages which are fully functional in protein synthesis. Several laboratories have recently reported the purification of phage-modified RNA polymerase from B. subtilis infected by SP01 (8, 16) or SP82 (17, 18), a closely related bacteriophage whose DNA also contains hydroxymethyluracil instead of thymine. The fact that these preparations have altered specificities was shown by analyzing the transcripts by competitive hybridization with phage DNA of the RNAs made in vivo. Further specificity was demonstrated by analyzing the transcripts formed from "rapid-start" initiation complexes (8), or by hybridization to separated single strands of phage DNA (16, 19). None of these techniques gives information about whether the RNA synthesized in vitro can act as a functional template for synthesis of phage proteins of the correct specificity. We detect no host sigma subunit in our preparation, whereas Duffy and Geiduschek (8) report that a substantial fraction of their enzyme purified from cells infected with SPO still contains sigma. Since we used a modification of their purification procedure it is difficult for us to account for this discrepancy. It is possible that we have lost some sigma factor during the high salt gel filtration step of the purification, although enzyme from uninfected cells still retains a substantial amount of sigma. It seems likely that the difference can be partly explained by the fact that our enzyme was isolated at 15 min after infection, whereas Duffy and Geiduschek harvested their infected cells at 10 min after infection, perhaps before modification of host enzyme was complete. Pero et al. purified their enzyme from infected cells using chromatography on phosphocellulose, a procedure which would have eliminated any sigma from the RNA polymerase preparation if it had been present (8). The results presented here clearly show that the positive control element for synthesis of functionally active mRNA for a phage middle enzyme copurifies with the phage-modified host RNA polymerase. In addition, the positive control probably operates at initiation rather than antitermination (Fig. 2). The fact that some mRNAs transcribed by B. subti-
Proc. Nat. Acad. Sci. USA 72 (1975)
lis RNA polymerase are not made by the SPOl-modified en-
zyme implies that the same modification may play a role in negative control of phage transcription.
Information obtained from studies of this sort must be considered together with data obtained by others using competitive hybridization. Since we only detect transcripts that can function in protein synthesis, we would not have detected nonspecific transcripts which do not contain ribosomal binding sites i.e., antimessage. However, transcription by SP01-modified RNA polymerase has been shown to be highly asymmetric (8, 16). Now that we know that the phagemodified RNA polymerase is sufficient to carry out in vitro synthesis of complete middle mRNA, the way is open to investigate the role of the newly associated polypeptides. Of particular interest will be the fate of the host sigma subunit, the negative control over synthesis of phage early messages (20), and the existence of phage-specific polypeptides which confer new promotor site selection. Duffy et al. have recently presented evidence that a phage protein of molecular weight 28,000 (perhaps equivalent to peptide VI in our preparation) is a positive control factor of SP01-modified RNA polymerase (21). We would like to thank Drs. Duffy and Geiduschek for a copy of
their manuscript (8) prior to publication. This project was supported by National Institute of Allergy and Infectious Diseases Research Grant no. Al 11323. U.S. Public Health Service/Department of Health, Education, and Welfare. 1.
2. 3. 4. 5. 6. 7. 8.
9.
10. 11. 12.
13. 14. 15. 16. 17. 18. 19. 20. 21.
Gage, L. P. & Geiduschek, E. P. (1971) J. Mol. Biol. 57, 279300. Fujita, D. J., Ohlsson-Wilhelm, B. M. & Geiduschek, E. P. (1971) J. Mol. Biol. 57,301-317. Okubo, S., Yanagida, T., Fujita, D. J. & Ohlsson-Wilhelm, B. M. (1972) Biken J. 15,81-97. Geiduschek, E. P. & Sklar, J. (1969) Nature 221,833-836. Duffy, J. J. & Geiduschek, E. P. (1973) FEBS Lett. 34, 172174. Chamberlin, M. J. (1974) Annu. Rev. Biochem. 43,721-775. Shub, D. A. (1975) Mol. Gen. Genet. 137, 171-180. Duffy, J. J. & Geiduschek, E. P. (1975) J. Biol. Chem. 250, 4530-4541. Bautz, E. K. F. & Dunn, J. J. (1971) in Procedures in Nucleic Acid Research, eds. Cantoni, G. L. & Davies, D. R. (Harper & Row, New York), Vol. 2, pp. 743-747. Studier, F. W. (1973) J. Mol. Biol. 79,237-248. Blattler, D. P., Garner, F., van Slyke, K. & Bradley, A. (1972) J. Chromatogr. 64, 147-155. O'Farrel, P. Z. & Gold, L. M. (1973) J. Biol. Chem. 248, 5502-5511. Laemmli, U. K. (1970) Nature 227,680-685. Shorenstein, R. G. & Losick, R. (1973) J. Biol. Chem. 248, 6163-6169. Geiduschek, E. P., Wilson, D. L. & Gage, L. P. (1969) J. Cell. Physiol. 74, Suppl. 1, 81-86. Pero, J., Nelson, J. & Fox, T. D. (1975) Proc. Nat. Acad. Sci. USA 72, 1589-1593. Spiegelman, G. B. & Whiteley, H. R. (1974) J. Biol. Chem. 249, 1476-1482. Spiegelman, G. B. & Whiteley, H. R. (1974) J. Biol. Chem. 249, 1483-1489. Lawrie, J. M., Spiegelman, G. B. & Whiteley, H. R. (1975) J. Virol. 15, 1286-1288. Gage, L. P. & Geiduschek, E. P. (1967) J. Mol. Biol. 30, 435440. Duffy, J. J., Petrusek, R. L. & Geiduschek, E. P. (1975) Proc. Nat. Acad. Sci. USA 72,2366-2370.
Vol. 72, No. 12, pp. 4886-4890, December 1975 Biochemistry
Synthesis of specific functional messenger RNA in vitro by phage-SPO1-modified RNA polymerase of Bacillus subtilis (protein synthesis in vitro/rifampicin)
MARSHAL SWANTON*, DONNA H. SMITH, AND DAVID A. SHUB Department of Biological Sciences, State University of New York at Albany, Albany, N.Y. 12222
Communicated by Cyrus Levinthal, September 30,1975
ABSTRACT RNA polymerase (nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) was purified from rifampicin-resistant Bacillus subtifis, from both uninfected cells and cells infected with bacteriophage SPOl. The enzyme from infected cells lacked all traces of the sigma subunit, contained several polypeptides absent from the enzyme made in uninfected cells, and had an altered template specificity in a transcription assay. A cell-free protein synthesizing system from Escherichia coli, when poisoned with rifampicin, was completely dependent on addition of either of these RNA polymerase preparations for DNA-dependent protein synthesis. Under these conditions, the SPOI-modified RNA polymerase preferentially stimulated the synthesis of functional mRNA for the phage enzyme dCMP deaminase (deoxycytidylate aminohydrolase, EC 3.5.4.12), whereas unmodified B. subtilis RNA polymerase could stimulate synthesis of this mRNA in small quantity and only after prolonged incubation. This mRNA belongs to a class of phage transcripts (m) which cannot be transcribed in vivo in the absence of phage-specific protein synthesis.
Competitive RNA-DNA hybridization is a very powerful tool which can be used to determine the relative amount of specific nucleotide sequences in a population of in vitro synthesized RNA molecules. However, it is much more difficult to obtain precise information about the details of the specificity of synthesis, i.e., do the in vitro synthesized RNA molecules start and end at the correct place?; is the fidelity of transcription high?; etc. These questions can best be answered by using as the assay for selective transcription the ability of the in vitro synthesized RNA molecules to function as the message for a specific protein in a coupled transcription-translation cell-free system (6). We have previously shown (7) that such an Escherichia coli cell-free system is capable of using SPOl DNA or RNA to stimulate very efficient protein synthesis. In this report, we show that purified, phage-modified Bacillus subtilis RNA polymerase transcribes SPO1 DNA in this system preferentially into RNA which functions as message for the middle (m) phage-specific enzyme dCMP deaminase (deoxycytidylate aminohydrolase, EC 3.5.4.12). In addition we show that B. subtilis and SP01-modified RNA polymerases transcribe a qualitatively different set of functional, SP01-specific, messenger RNAs in this system.
Transcription of the genome of bacteriophage SP01 has been extensively studied by competitive RNA-DNA hybridization (1). Six classes of transcripts have been described, which are independently controlled with respect to the turning on or turning off of their synthesis. Two of these (e and em) are synthesized after infection without detectable lag, and are subsequently repressed if phage protein synthesis is allowed to occur. Three additional classes of RNA (m, mll and m21) are made independently of phage DNA replication, but require prior phage protein synthesis. In particular, the protein product of cistron 28 is required for m and m1l expression, and the protein products of cistrons 33 and 34 are required for M21 RNA synthesis (2, 3). After phage DNA synthesis begins, another population of transcripts (1) is made. The existence of three pre-replicative classes of phagespecific transcripts (m, m1l, and M21), whose synthesis is under phage genetic control, suggested the possibility that mature phage DNA extracted from purified virus might be an appropriate template for a new RNA polymerase (nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) of the correct specificity for these classes. Since it had been shown that the rifampicin sensitivity of RNA synthesis throughout SP01 infection was determined by the sensitivity of the host's RNA polymerase (4) it was logical to expect that the new specificity for middle RNA synthesis should reside in the host's RNA polymerase, which had been modified in some way. Duffy and Geiduschek (5) demonstrated that the expected in vitro selectivity for phage middle transcripts does, indeed, reside in an enzyme which contains the host core and other peptides which are made after phage infection. *
MATERIALS AND METHODS Bacteria and Phage. Bacillus subtilis 1005 (rifR), a derivative of W168 resistant to high levels of rifampicin, was obtained from Dr. T. Leighton. F21, a suppressible nonsense mutant of phage SP01, and its permissive host B. subtilis HAlOiB, were obtained from Dr. E. P. Geiduschek. Isolation of RNA Polymerase. B. subtilis 1005 was grown in 12 liter batches in a New Brunswick fermentor. The medium (2XLB) contains (weight/volume): 2.5% Bacto tryptone (Difco), 2.0% yeast extract (Difco), 0.3% NaH2PO4, and 3% glucose. This medium supports logarithmic growth (with vigorous aeration) to an OD540 of greater than 2. Cultures were harvested at OD540 of 1.5 or infected with SP01 (multiplicity of infection = 5.0) at OD540 of 1.0 and harvested after 15 min. The protease inhibitor phenylmethylsulfonyl fluoride was added at 0.5 mM to uninfected cultures 10 min before harvest and to infected cultures 5 min before harvest. All cultures were harvested by pouring over ice and collected in a refrigerated continuous flow centrifuge. Cells were washed once with buffer I [10 mM Tris-HCl (pH 8.0), 50 mM KC1, 10 mM MgCl2, 0.1 mM dithiothreitol, 0.1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10% (vol/vol) glycerol] and stored in liquid N2. We used a modification of the procedure of Duffy and Geiduschek (8) for enzyme purification. All operations were performed at 40. Cell pellets (20 g or more) were resuspended in 2 volumes of buffer I, homogenized in a Sorvall omnimixer, and passed through a French pressure cell at 10-
Present address: Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colo. 80302.
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Table 1. Purification of RNA polymerase of B. subtilis Uninfected cells (20.3 g)
Crude lysate High-speed supernatant Gel filtration DEAE-cellulose DNA-cellulose
SPO1-infected cells (31.7 g)
Total units
% Recovery
288
100
803
100
300 178 84 54
104 62 29 19
908 482 242 146
113 60 30 18
Vi.--l--
Total % units Recovery
_-VII
_._
-VI -IV
RNA polymerase activity was determined after various stages of purification, described in Materials and Methods. Activity is expressed as units/g of packed cells.
-111
-1I12,000 pounds/inch2 (69-83 MPa). Debris was removed at 20,000 X g for 30 min. Solid (NH4)2SO4 was added to the supernatant ("crude lysate") to 25% saturation and a highspeed supernatant was obtained by centrifugation at 136,000 X g for 3 hr. This supernatant was brought to 70% saturation with solid (NH4)2SO4; the precipitate was collected by centrifugation at 48,000 X g for 30 min, dissolved in 15-20 ml of buffer II [10 mM Tris-HCl (pH 8.0), 1.0 M KCl, 0.1 mM dithiothreitol, 0.1 mM EDTA, 15% (vol/vol) glycerol], and applied to a Bio-Gel A-1.5m agarose column (2.5 X 74 cm) equilibrated with buffer II. The column was eluted at 30 ml/hr and 5 ml fractions were collected. Active fractions were pooled, dialyzed against buffer II (without KCl) to reduce KC1 concentration, and precipitated by adding solid (NH4)2SO4 to 70% saturation. The precipitate was collected by centrifugation, resuspended in 15-20 ml of buffer III [10 mM Tris-HCl (pH 8.0), 50 mM KCI, 0.1 mM dithiothreitol, 1 mM EDTA, 15% (vol/vol) glycerol] and applied to a DEAEcellulose (Whatman DE-52) column (2.5 X 11 cm) equilibrated with buffer III. The RNA polymerase activity was eluted with a linear 0.05-0.5 M KCI gradient at 50 ml/hr. Active fractions were pooled, diluted to less than 0.15 M KCI, and applied to a calf thymus DNA (Sigma, type I) column (2 X 11 cm) containing 350 ,ug of DNA per packed ml (9). The column was equilibrated with buffer III (containing 0.15 M KCI) and RNA polymerase activity was eluted with a linear 0.15-1.0 M KCI gradient. Active fractions were pooled, concentrated in an Amicon ultra filtration device using a UM10 filter, dialyzed against storage buffer [10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.1 mM dithiothreitol, 50% (vol/vol) glycerol] and stored at -200. Enzyme Assays. RNA polymerase was assayed in 0.1 ml reaction mixtures containing: ATP, GTP, CTP, UTP, 1 mM each; 50 mM Tris-HCl, pH 8.0; 50 mM KCI; 10 mM MgCI2; 0.5 mM dithiothreitol; [5-3H]UTP, 5 ,uCi/jimol; SP01 DNA, 50 jig/ml. Reaction mixtures were assembled on ice and started by warming to 370. Reactions were terminated after 10 min by precipitating with cold 5% CI3CCOOH, filtered, and counted in a scintillation counter. One unit is defined as incorporation of 1 nmol of UTP in 10 min. dCMP deaminase was assayed as previously described (7). Phage Nucleic Acid. Phage SP01, its mutant sus F21, and E. coli phage T4D (the gift of Drs. R. Trimble and F. Maley) were purified on a CsCI step gradient and DNA was extracted with phenol as previously described (7). RNA was prepared from B. subtilis harvested 10 min after phage infection as previously described (7). Gel Electrophoresis. Sodium dodecyl sulfate polyacrylamide slab gels were prepared (10) with 15% acrylamide
A B C
D E F
FIG. 1. Sodium dodecyl sulfate gel electrophoresis of RNA polymerase. Sodium dodecyl sulfate acrylamide gels (15%) were prepared using the buffer system of Laemmli (13). Enzyme from SP01-infected B. subtilis, A: 1.74 units; B: 0.87 units; C: 0.43 units. Enzyme from uninfected B. subtilis, D: 1.95 units; E: 0.98 units; F: 0.49 units.
and bisacrylamide/acrylamide = 0.0058 (11). Gels were run for 4-5 hr, fixed and stained in: 50% (vol/vol) methanol, 7% acetic acid, and 0.2% Coomassie brilliant blue, and destained in methanol, acetic acid, water (5:1:5). Stain intensity was determined by scanning the gels with a Gilford spectrophotometer at 540 nm. Areas under peaks were cut out and weighed. In Vitro Protein Synthesis. Conditions were as previously described (7, 12) except that, when purified B. subtilis RNA polymerase was used to stimulate protein synthesis, the enzyme and DNA were incubated with triphosphates, separately for 2 min at 370 before adding the other components of the cell-free protein synthesis system. Incubations were at 370 for an additional 30 min. Protein synthesis was followed by incorporation of [2-14C]leucine (54 gCi/Asmol) into hot CL6CCOOH-insoluble form (7). at 100-125 V
RESULTS DNA-dependent RNA polymerase was purified from phageSPOl-infected or uninfected B. subtilis as described in Materials and Methods. The two preparations behaved identically at each step of the purification. The total units and percent recovery at each step are shown in Table 1. Specific activities were 430 units/mg for the B. subtilis enzyme and 270 units/mg for the enzyme prepared from infected cells. The enzyme preparation obtained after DNA-cellulose chromatography was subjected to sodium dodecyl sulfateacrylamide gel electrophoresis (Fig. 1). The gels were scanned and the relative concentrations of the principal polypeptides were determined (Table 2). Molecular weights of these polypeptides were approximated by comparing their mobilities with a set of proteins of known molecular weight. These are only estimates, since it is known (13) that this gel system can give incorrect molecular weight values. In addition to the polypeptides of B. subtilis core RNA polymerase ( alj3',a), we observe four additional polypeptides in both the uninfected and SPOl-infected enzyme preparations. One of these (peptide VIII, molecular weight 110,000) is present in much higher amount in the SPOl-infected enzyme, and a polypeptide of this molecular weight has been reported as a
Proc. Nat. Acad. Sci. USA 72 (1975)
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Table 2. Relative concentration and molecular weights of polypeptides associated with B. subtilis and SP01-modified RNA polymerase
Table 3. Template specificity of RNA polymerase Relative activity DNA
Concentration relative to a2 PP
Peptide
Uninfected
Sigma I II III IV V VI VII VIII
0.33 0.48 0.77
0.35 0.05
Uninfected
SP01-modified
1.0 0.99 0.47
1.0 0.10 0.28
SPOl
SP01infected
Molecular weight
1.11 1.37 0.45 0.15 0.37 1.02 0.93 0.45
11,000 12,500 17,000 19,500 23,500 26,000 47,000 110,000
The sodium dodecyl sulfate-acrylamide gel from Fig. 1 was scanned and areas under peaks were quantitated. Relative molar concentrations were normalized to the molar concentration of a2O3'. Molecular weights were estimated using standards: bovine serum albumin (68,000), glutamate dehydrogenase (53,000), gamma globulin heavy chain (55,000), yeast alcohol dehydrogenase (37,000), chymotrypsinogen (25,700), and myoglobin (17,200).
contaminant of B. subtilis RNA polymerase purified on DNA-cellulose columns (14). The other three peptides resemble new subunits recently found in B. subtilis RNA polymerase (8, 16). The RNA polymerase preparation from SP01-infected cells differs from the uninfected enzyme in several ways. There is no detectable sigma subunit in the SPO-infected enzyme, even though enzyme prepared by identical methods from uninfected cells yields a large amount of sigma. There are, however, four new polypeptides unique to the enzyme prepared from SPO-infected cells. One of them (peptide IV) is present in a low molar ratio. The others (peptides III, VI, and VII) are present in sufficiently high molar ratio to be likely candidates for new phage-specified enzyme subunits. New polypeptides associated with purified SP01- (or SP82-) modified RNA polymerase have been reported (8, 16, 17). Several of these have molecular weights similar to our peptides III, IV, and VI. A polypeptide similar in size to peptide VII has not previously been reported as a component of highly purified phagemodified enzyme. One can see, from Table 3, that the SP01-modified RNA
T4 Calf thymus
Uninfected RNA polymerase (19.5 units/ml) and SP01-modified RNA polymerase (17.4 units/ml) were assayed as described in Materials and Methods, using several templates at DNA excess. Activities are expressed relative to SP01 DNA.
polymerase has a dramatically different template-specificity than the B. subtilis enzyme. Whereas both phage SP01 and T4 DNAs are excellent templates for the enzyme from uninfected cells, the SP01-modified enzyme retains high activity on SP01 DNA, but has virtually lost the ability to transcribe T4 DNA. Both enzymes are active on calf thymus DNA although at reduced levels compared to SP01 DNA. Our objectives in this work were to determine: (1) if RNA synthesized by SP01-modified RNA polymerase could function in protein synthesis, (2) whether these proteins were complete and functional (enzymatically active) and, (3) whether the proteins were of the correct specificity (middle). Table 4 shows that when a cell-free DNA-dependent protein synthesizing system is poisoned with the RNA polymerase inhibitor rifampicin overall protein and enzyme (dCMP deaminase) synthesis fall to very low levels. These levels can be restored by adding RNA polymerase from uninfected B. subtilis which is resistant to rifampicin. However, a dramatic increase in dCMP deaminase synthesis, over that of the standard cell free system, is observed when the added RNA polymerase is SP01-modified. This preferential stimulation of synthesis of dCMP deaminase by SP01modified RNA polymerase was encouraging, since dCMP deaminase has been shown to be an enzyme under middle (m) mRNA control (7). The SP01 mutant sus F21 (cistron 28) is defective in transcription of middle messages (2) and does not make dCMP deaminase in nonpermissive infections (3). DNA from sus F21 is as competent as wild-type SP01 DNA in directing synthesis of dCMP deaminase mRNA by the B. subtilis or SP01-modified RNA polymerase (Table 4). This shows that the in vitro synthesized product of gene 28 is not acting in vitro in the synthesis of dCMP deaminase message by either enzyme.
Table 4. In vitro protein synthesis stimulated by uninfected or SP01-modified B. subtilis RNA polymerase
Rifampicin
Template SP01 DNA SP01 DNA SP01 DNA SP01 DNA F21 DNA F21 DNA 10 min RNA
RNA polymerase
10
jig/ml +
SP01-infected Uninfected SP01-infected Uninfected
+ + + + + +
SP01-infected
+
Uninfected
+
dCMP deaminase
(units/S50 l) 244 0 1270 228 1536 196 1846 8 46 0
Leucine incorporation (pmol/50, l) 1327 73 2050 1002 2040 948 669 101
Cell-free protein synthesis was carried out as described in Materials and Methods. Concentrations of added template were: SP01 DNA, 45 Mg/ml; F21 DNA, 35 Mg/ml; RNA extracted from SP01-infected cells at 10 min after infection, 1.2 mg/ml. RNA polymerase from uninfected B. subtilis was added at 19.5 units/ml and SP01-modified RNA polymerase was added at 17.4 units/ml. In determining dCMP deaminase activity, the enzyme blank, equivalent to 54 units/50 Ml, has been subtracted from all values.
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Proc. Nat. Acad. Sci. USA 72 (1975)
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.
X----X
c
x
500 x
x d
-6
0
-
I
10 Minutes
20
FIG. 2. Time course of synthesis of dCMP deaminase in vitro. In vitro protein synthesis reactions were programmed with SP01 DNA (45 gtg/ml). Uninhibited cell-free system, no addition (0); plus rifampicin (10 gg/ml) and B. subtilis RNA polymerase (-); plus rifampicin (10 gg/ml) and SP01-modified RNA polymerase (Xi. At various times after starting the reactions, 50 gl samples were removed into actinomycin (10 ug/ml) and incubation was continued for a total of 30 min. dCMP deaminase activity was determined and expressed as units/50 Al.
If SPOI-modified RNA polymerase initiates transcription at middle promotors of SP01 DNA, one would expect dCMP deaminase to appear very rapidly in protein synthesis incubations stimulated by this polymerase. On the other hand, if dCMP deaminase synthesis in reactions stimulated by uninfected B. subtilis RNA polymerase is the result of readthrough by this enzyme, then there should be a longer lag in its appearance. The experiment described in Fig. 2 confirms these expectations and supports our conclusions that the SP01-modified RNA polymerase we have prepared exhibits selectivity for middle phage promotors. To gain a more general view of transcription specificity of these two RNA polymerase preparations, cell-free protein synthesis was carried out with [2-14C]leucine. Reactions mixtures were separated by gel electrophoresis and radioactive proteins were detected by radioautography. Fig. 3 shows that both enzyme preparations direct the synthesis of proteins of discrete molecular weights. Several proteins appear to be synthesized in response to both RNA polymerases, but there are other proteins whose mRNA is a product of one of the enzymes, and not of the other. The SPO-modified RNA polymerase exhibits, therefore, both the positive and negative control functions required during the middle phase of SP01 development. A fuller characterization of the radioactive proteins displayed in Fig. 3 will be presented elsewhere.
DISCUSSION We have purified RNA polymerase from B. subtilis infected by phage SPOl using standard techniques. Our enzyme preparation contains all the polypeptides present in the host cell RNA polymerase, with the exception of the sigma subunit. There are, in addition, three new polypeptides which copurify with the phage enzyme. The results of Table 3 show that the phage-modified enzyme has an altered template specificity. Although the phage-modified RNA polymerase is even more active than host enzyme on SPOl DNA, it is virtually inactive when T4 DNA is the template. RNA polymerases of E. coli and B. subtilis are both' extremely active on the SP01 DNA template and both make identical populations of transcripts, whether assayed by competition hybridization (15) or by gel electrophoresis of polypeptides synthesized in the coupled transcription-translation system used in this work (in preparation). Since the SPO-phage-modified RNA polymerase retains high activity on SP01 DNA, the loss of activity on the
3 1 2 FIG. 3. Gel electrophoresis of SP01 proteins synthesized in vitro. Reactions were carried out with SP01 DNA and [2-'4C]leucine as described in Materials and Methods and the legend to Table 4. After 30 min, samples were mixed with an equal volume of 2-fold concentrated "final sample buffer" of Laemmli (13) and heated for 1 min at 900. Gel electrophoresis was carried out as described in Materials and Methods. After destaining the gels were dried (10) and exposed for radioautography with Kodak No Screen x-ray film. Columns 2 and 3 each represent duplicate reactions. (1) E. coli cell-free system plus rifampicin. (2) Plus SP01-modified RNA polymerase, 17.4 units/mL (3) Plus B. subtilis RNA polymerase, 19.5 units/ml.
T4 DNA template indicates that this enzyme has an altered promotor specificity. B. subtilis holoenzyme is equally active with SPO or T4 DNA, and the lack of activity of SP01modified enzyme on T4 DNA could be a consequence of the loss of sigma factor. In fact, it has been shown that the behavior of SP01-modified RNA polymerase on heterologous template is similar to that of core polymerase, including the ability to be stimulated by B. subtilis sigma factor (8). Our previous work (7) has shown that the phage-induced enzyme dCMP deaminase is a protein whose mRNA is middle (m) in the terminology of Gage and Geiduschek (1). That is, transcription of mRNA for dCMP deaminase is initiated only after the third minute of infection, reaches a maximum level in the cell at about the 10th minute, declines rapidly after the 13th minute, and is absolutely dependent on phage protein synthesis (7). A coupled transcriptiontranslation system system (12) from E. coli could synthesize dCMP deaminase when primed with SPOl DNA, but only after a long lag and at relatively low levels (7). Since the RNA initiations which ultimately led to dCMP deaminase synthesis occurred without lag, we interpreted the synthesis of dCMP deaminase mRNA as resulting from read-through transcription by the E. coli RNA polymerase. In the present work, we have continued to utilize the E. coli cell-free system for translation, but have poisoned the endogenous E. coli RNA polymerase with rifampicin, making DNA-dependent protein synthesis entirely dependent on addition of purified RNA polymerase extracted from rifampicin-resistant B. subtilis. Our expectation was that addition of RNA polymerase prepared from vegetative cells of B. subtilis would produce a reconstituted system identical to the uninhibited E. coil system with regard to dCMP deaminase synthesis. On the other hand, if the SPO-phage-modified RNA polymerase had retained its' promotor specificity in vitro, we expected to observe synthesis and translation of .dCMP deaminase mRNA without lag and at higher levels. The results of our experiments have confirmed our predictions. Using SP01 DNA as a template, the uninhibited E. coli cell-free system synthesizes some dCMP deaminase
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after a lag of 10 min, in agreement with our previous results (7). When E. coli RNA polymerase is poisoned by rifampicin, addition of RNA polymerase from uninfected B. subtilis restores protein synthesis to a high level, but allows the synthesis of barely detectable levels of dCMP deaminase (Table 4). The amount of enzyme ultimately produced is so low that we are unable to extrapolate an initial time of appearance; the earliest time at which enzyme was detected was 9 min (Fig. 2). The amino-acid incorporation shows no lag and produces the same set of polypeptides of discrete molecular weights as does the uninhibited cell-free system (manuscript in preparation). The virtual absence of transcription of dCMP deaminase mRNA may be due to higher fidelity in termination of the B. subtilis RNA polymerase than the E. coli enzyme. However, addition of SPOI-modified RNA polymerase to the rifampicin-poisoned cell-free system stimulates protein synthesis and dCMP deaminase synthesis to high levels (Table 4), also without any appreciable lag (Fig. 2). When the radioactive proteins made in this system are analyzed by gel electrophoresis, they appear to be a completely different population from those made in response to stimulation by RNA polymerase from uninfected cells (Fig. 3). Therefore, we conclude that SPOI-modified RNA polymerase exhibits the same alteration of promotor selectivity in vitro as it does in vivo, and that much of the subsequent transcription is of messages which are fully functional in protein synthesis. Several laboratories have recently reported the purification of phage-modified RNA polymerase from B. subtilis infected by SP01 (8, 16) or SP82 (17, 18), a closely related bacteriophage whose DNA also contains hydroxymethyluracil instead of thymine. The fact that these preparations have altered specificities was shown by analyzing the transcripts by competitive hybridization with phage DNA of the RNAs made in vivo. Further specificity was demonstrated by analyzing the transcripts formed from "rapid-start" initiation complexes (8), or by hybridization to separated single strands of phage DNA (16, 19). None of these techniques gives information about whether the RNA synthesized in vitro can act as a functional template for synthesis of phage proteins of the correct specificity. We detect no host sigma subunit in our preparation, whereas Duffy and Geiduschek (8) report that a substantial fraction of their enzyme purified from cells infected with SPO still contains sigma. Since we used a modification of their purification procedure it is difficult for us to account for this discrepancy. It is possible that we have lost some sigma factor during the high salt gel filtration step of the purification, although enzyme from uninfected cells still retains a substantial amount of sigma. It seems likely that the difference can be partly explained by the fact that our enzyme was isolated at 15 min after infection, whereas Duffy and Geiduschek harvested their infected cells at 10 min after infection, perhaps before modification of host enzyme was complete. Pero et al. purified their enzyme from infected cells using chromatography on phosphocellulose, a procedure which would have eliminated any sigma from the RNA polymerase preparation if it had been present (8). The results presented here clearly show that the positive control element for synthesis of functionally active mRNA for a phage middle enzyme copurifies with the phage-modified host RNA polymerase. In addition, the positive control probably operates at initiation rather than antitermination (Fig. 2). The fact that some mRNAs transcribed by B. subti-
Proc. Nat. Acad. Sci. USA 72 (1975)
lis RNA polymerase are not made by the SPOl-modified en-
zyme implies that the same modification may play a role in negative control of phage transcription.
Information obtained from studies of this sort must be considered together with data obtained by others using competitive hybridization. Since we only detect transcripts that can function in protein synthesis, we would not have detected nonspecific transcripts which do not contain ribosomal binding sites i.e., antimessage. However, transcription by SP01-modified RNA polymerase has been shown to be highly asymmetric (8, 16). Now that we know that the phagemodified RNA polymerase is sufficient to carry out in vitro synthesis of complete middle mRNA, the way is open to investigate the role of the newly associated polypeptides. Of particular interest will be the fate of the host sigma subunit, the negative control over synthesis of phage early messages (20), and the existence of phage-specific polypeptides which confer new promotor site selection. Duffy et al. have recently presented evidence that a phage protein of molecular weight 28,000 (perhaps equivalent to peptide VI in our preparation) is a positive control factor of SP01-modified RNA polymerase (21). We would like to thank Drs. Duffy and Geiduschek for a copy of
their manuscript (8) prior to publication. This project was supported by National Institute of Allergy and Infectious Diseases Research Grant no. Al 11323. U.S. Public Health Service/Department of Health, Education, and Welfare. 1.
2. 3. 4. 5. 6. 7. 8.
9.
10. 11. 12.
13. 14. 15. 16. 17. 18. 19. 20. 21.
Gage, L. P. & Geiduschek, E. P. (1971) J. Mol. Biol. 57, 279300. Fujita, D. J., Ohlsson-Wilhelm, B. M. & Geiduschek, E. P. (1971) J. Mol. Biol. 57,301-317. Okubo, S., Yanagida, T., Fujita, D. J. & Ohlsson-Wilhelm, B. M. (1972) Biken J. 15,81-97. Geiduschek, E. P. & Sklar, J. (1969) Nature 221,833-836. Duffy, J. J. & Geiduschek, E. P. (1973) FEBS Lett. 34, 172174. Chamberlin, M. J. (1974) Annu. Rev. Biochem. 43,721-775. Shub, D. A. (1975) Mol. Gen. Genet. 137, 171-180. Duffy, J. J. & Geiduschek, E. P. (1975) J. Biol. Chem. 250, 4530-4541. Bautz, E. K. F. & Dunn, J. J. (1971) in Procedures in Nucleic Acid Research, eds. Cantoni, G. L. & Davies, D. R. (Harper & Row, New York), Vol. 2, pp. 743-747. Studier, F. W. (1973) J. Mol. Biol. 79,237-248. Blattler, D. P., Garner, F., van Slyke, K. & Bradley, A. (1972) J. Chromatogr. 64, 147-155. O'Farrel, P. Z. & Gold, L. M. (1973) J. Biol. Chem. 248, 5502-5511. Laemmli, U. K. (1970) Nature 227,680-685. Shorenstein, R. G. & Losick, R. (1973) J. Biol. Chem. 248, 6163-6169. Geiduschek, E. P., Wilson, D. L. & Gage, L. P. (1969) J. Cell. Physiol. 74, Suppl. 1, 81-86. Pero, J., Nelson, J. & Fox, T. D. (1975) Proc. Nat. Acad. Sci. USA 72, 1589-1593. Spiegelman, G. B. & Whiteley, H. R. (1974) J. Biol. Chem. 249, 1476-1482. Spiegelman, G. B. & Whiteley, H. R. (1974) J. Biol. Chem. 249, 1483-1489. Lawrie, J. M., Spiegelman, G. B. & Whiteley, H. R. (1975) J. Virol. 15, 1286-1288. Gage, L. P. & Geiduschek, E. P. (1967) J. Mol. Biol. 30, 435440. Duffy, J. J., Petrusek, R. L. & Geiduschek, E. P. (1975) Proc. Nat. Acad. Sci. USA 72,2366-2370.
