Proc. Nat. Acad. Sci. USA Vol. 70, No. 10, pp. 2845-2849, October 1973

Modification of RNA Polymerase after T3 Phage Infection of B (DEAE-cellulose, phosphocellulose, and DNA-cellulose chromatography/h' subunit)

B. DHARMGRONGARTAMA, S. P. MAHADIK, AND P. R. SRINIVASAN Columbia University, College of Physicians & Surgeons, Department of Biochemistry, New York, N. Y. 10032 Communicated by Erwin Chargaff, June 25, 1973

ABSTRACT E. coli B cells infected with T3 phage con- 0.15 M KCl, 125,ug of bovine-serum albumin, 25 ug of calf- tain a modified host RNA polymerase in addition to the thymus DNA or E. coli DNA or 10 of either T2 or T3 normal RNA polymerase found in uninfected cells. The /Ag modified RNA polymerase behaves differently in its elution DNA, 0.2 mM "4C-labeled (ATP or properties from the normal enzyme on DEAE-cellulose, GTP, 1 Ci/mol), and 0.2 mM (each) of the other three un- phosphocellulose, and DNA-cellulose column chroma- labeled triphosphates, and enzyme. Incubation was at 370 for tography. The modified enzyme also differs from the 10 min. The specific activity is expressed as units per mg of normal polymerase in some of its enzymatic parameters. 1 of incorporated into The specific activity of the modified RNA polymerase is protein; one unit equals nimol [14C]GTP markedly lower (i.e., 1/4) than that of the normal enzyme. RNA in 10 min at 370 with native calf-thymus DNA as The decrease in activity is probably due to an alteration in template. the fl' subunit of the polymerase. A similar modification is also observed in nonpermissive cells infected with a Na Dodecyl Sulfate-Polyacrylamide-Gel Electrophoresis of -i amber mutant incapable of producing active T3- Labeled Polymerases. E. coli B was grown to a cell density of specific T3 polymerase. An analogous modification in host 5 X 108 cells per ml at 370 and then shifted to 300. After 15 RNA polymerase does not seem to occur in E. coli cells min the culture was divided into four equal portions of 20 ml infected with T7 phage. each. One culture was used as a control, and the other three The mechanisms by which an invading virulent can cultures received wild-type T3 phage or T3 amH5 (an amber arrest the macromolecular synthesis of its host and initiate mutant in gene 1) or T7 phage. 2 min later, 100 uCi of "IC- synthesis of phage-specific proteins are being studied in several labeled reconstituted protein hydrolysate (Schwarz mixture, phage-infected systems (1-3). We have previously reported Schwarz BioResearch) was added to each flask. After 8 min of the isolation of an inhibitory protein of Escherichia coli RNA incorporation, the cells were rapidly chilled and harvested by polymerase from E. coli B cells infected with T3 phage, which centrifugation. For polymerase isolation, each sample was may be responsible for the arrest of host RNA synthesis (4). mixed with 1 g of unlabeled E. coli B cells before the cell-free During that study we observed that the total activity of host extract was subjected to (NH4)2SO4 fractionation. All solu- RNA polymerase from T3-infected cells freed from the in- tions contained phenylmethylsulfonylfluoride (0.25 mg/ml) to hibitory protein and nucleases was considerably lower than the inhibit proteases. Sufficient rabbit antiserum prepared against activity of RNA polymerase isolated from uninfected cells. We modified RNA polymerase was added to the (NH4)2SO4 present evidence here that suggests that this decrease in fraction to precipitate 20,g of RNA polymerase. After 18 hr activity is due to a structural modification of the host RNA at 40, the precipitate was collected, washed extensively with polymerase, which is accompanied by various alterations in its saline, and finally with H20. After it was dried briefly in a enzymatic properties. desiccator over Drierite, the precipitate was dissolved in 0.01 M phosphate buffer (pH 7.2) containing 1% Na dodecyl METHODS sulfate, 0.5 M mercaptoethanol, and 10% glycerol, by heating Preparation of Polymerases from Normal and Infected Cells. in a boiling-water bath for 10 min. Na dodecyl sulfate-gel RNA polymerase holoenzyme of E. coli B was prepared by the electrophoresis was done by published methods (7, 8). At the method of Burgess by ammonium sulfate fractionation and end of the run, the gels were stained by Coomassie brilliant DEAE-cellulose chromatography (5). The enzyme was blue to detect the protein bands. After they were destained, further purified by chromatography on DNA-cellulose col- the gels were cut into 1-mm slices and solubilized by incuba- umns (6), or by high-salt and low-salt glycerol gradient tion at 370 for 16 hr with Omnifluor-toluene scintillation fluid centrifugation (5) and then by DNA-cellulose column chroma- containing 3% Protosol (New England Nuclear Corp.). tography. Core polymerase was prepared from the holoenzyme by chromatography on phosphocellulose (5). RESULTS The modified RNA polymerase was isolated from E. coli B Altered Behavior of Polymerase of E cells infected at with T3 at a multiplicity of 5-10 as Chromatographic PNA 300 phage B column described (4). 10 min after infection, the cells were rapidly coli from T3-Infected Cells. DEAE-cellulose of from E. B with chilled to 40 and harvested by centrifugation. chromatography extracts coli cells infected phage T3 resolves the host RNA polymerase activity into RNA Polymerase Assay. The reaction mixture for RNA two fractions, A and B (Fig. 1). Fraction A elutes near 0.2 M synthesis contained in 0.25 ml: 40 mM Tris * HC1 buffer (pH KCl, the same salt concentration required to elute normal 7.9), 10 mM MgCl2, 0.1 mM EDTA, 0.1 mM dithiothreitol, RNA polymerase from uninfected cells. A second peak of 2845 Downloaded by guest on October 2, 2021 2846 Cell Biology: Dharmgrongartama et al. Proc. Nat. Acad. Sci. USA 70 (1973)

cmi 0 x E 0~ 00. 0- 0 0 0 o 0 0.C) t2- 02aC 0 4 8 12 16 20 Enzyme (g) Fraction Number FIG. 2. Variation of RNA polymerase activity with protein FIG. 1. DEAE-cellulose chromatography of RNA polymerase concentrations. The standard assay mixture contained the from T3-infected E. coli B cells. RNA polymerase was purified indicated amount of enzyme and 25 ug of calf-thymus DNA. from T3-infected cells by the method of Burgess up to the high- Concentration of protein was estimated by the method of Lowry speed supernatant and then subjected to ammonium sulfate et al. (14). 0, Normal holoenzyme, specific activity = 937; fractionation. The fraction precipitating between 33 and 70% 0, modified holoenzyme, specific activity = 225. saturation was dissolved in buffer A [10 mM Tris' HOl (pH 7.9)- 10 mM MgCl2-0.1 mM EDTA-0.1 mM dithiothreitol-5% on DNA-cellulose columns; its properties were compared with glycerol], loaded onto a DEAE-cellulose column, and washed those of normal RNA polymerase. From the rate of RNA with buffer A followed by a 0-0.5 M KCl gradient in the same synthesis with increasing concentration of enzyme (Fig. 2), it buffer. Aliquots from the'fractions were assayed for AMP in- is clear that the specific activity of the modified enzyme is one- corporation with calf-thymus DNA as template in standard fourth that of the normal enzyme. Both enzymes have identi- reaction mixtures. *, 14C; 0, A280. cal heat stability. They are equally sensitive to rifampicin inhibition when assayed with E. coli DNA, calf-thymus DNA, activity, fraction B, elutes at a higher salt concentration, 0.34 or T3 DNA templates. They have similar salt optima when M KCl; when fractions from this peak are chromatographed assayed with T3 DNA (0.1 M KCl) and E. coli DNA (0.1 and separately on another DEAE-cellulose column, this activity 0.3 M KC1). However, with calf-thymus DNA, modified RNA again elutes at 0.34 M 0KC1. The altered RNA polymerase polymerase has no salt requirement (Fig. 3), unlike the normal from T3-infected cells also behaves differently from normal RNA polymerase, which is most active at 0.05 M KCl. When RNA polymerase on phosphocellulose columns (Table 1). normal RNA polymerase is inhibited 50% by 0.34 M KCl the Whereas normal core polymerase can be eluted from phospho- modified enzyme is inhibited only 15%; and when the former cellulose columns at 0.33 M KCl (5), the altered enzyme is inhibited 97% by 0.4 M KCl, the activity of the modified requires higher salt concentration for its removal. Yet another enzyme is decreased by only 25%. Both enzymes can use difference in the chromatographic behavior of the modified either Mg++ or Mn++, but they require different concentra- RNA polymerase is its requirement of 0.58 M NaCl for elution tions for optimal activity. Normal RNA polymerase has a from DNA-cellulose columns, unlike the normal RNA single Mg++ optimum at 8 mM; modified enzyme has two polymerase, which can be eluted with 0.36 M NaCl. These optima, one at 4 mM and the other at 9 mM. Similarly, the results suggest that some component of the host RNA former is maximally active with 3 mM Mn++ while the latter polymerase is modified after T3 infection. enzyme again shows two optima, 2 mM and 6.5 mM. Neither Properties of Normal and Modified RNA Polymerase. The modified core enzyme nor modified holoenzyme inhibits fraction that elutes late from DEAE-cellulose columns (i.e., normal RNA polymerase. Modified core enzyme is capable of fraction B) free from the normal RNA polymerase was further using normal sigma subunit liberated from normal holoenzyme purified on low-salt and high-salt glycerol gradients or directly during RNA synthesis with T2 or T3 DNA templates (Table 2). TABLE 1. Elution position of various RNA polymerase Structural Differences Between Modified and Normal RNA preparations from DEAE-cellulose, phosphocellulose, and of the column Polymerases. The approximate molecular weights calf-thymus DNA-cellulose chromatography modified core and holoenzymes were estimated from their sedimentation values obtained from velocity sedimentation in of KCl or NaCl Molarity sucrose gradients. Normal core and holopolymerases were Enzyme DEAE- Phospho- DNA- used as external standards in parallel gradients. Catalase was preparation cellulose cellulose cellulose* the internal standard in all tubes. The sedimentation values Normal holoenzyme ^0. 18-0 .22 - 0.36 for the various enzymes in Table 3 were calculated using 12.5 Normal core enzyme - 0.33 0.46 S and 14.5 S as the sedimentation values and 400,000 and Modified holoenzyme 0.30-0.37 0.58 495,000 as the molecular weights of normal core polymerase Modified core enzyme 0.42-0.59 0.50 and holopolymerase, respectively (1). Compared to normal core enzyme, normal holoenzyme * NaCl was used for elution. sediments at 14.4 S, in good agreement with the published Downloaded by guest on October 2, 2021 Proc. Nat. Acad. Sci. USA 70 (1978) E. coli RNA Polymerase and T3 Phage Infection 2847 TABLE 2. Utilization of normal sigma factor by modified core enzyme 1.6 DNA template Activity E C 1L4 Calf ratio % Stimu- 1.2 Enzyme thymus T2/ T3/ lation co 0. preparation (CT) T2 T3 CT CT T2 T3 0 I 1. Normalholo- C 0.8 enzyme (A) 382 771 1484 - a- 2. Modified core 0.6 enzyme (B) 280 229 238 3. Normal holo- 0o.4 enzyme plus 0. modified core enzyme 533 1386 3168 2.70 5.92 270 706 4. Calculated 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 KCI concentration (M) value (sum ofA and B) 662 1000 1722 1.5 2;52 - FIG. 3. Salt requirement of normal (0) and modified (0) holoenzyme. The standard reaction mixture contained either 1.6 jAg of normal holoenzyme or 4.8 jug of modified holoenzyme with Holoenzyme (2 jg) was incubated for 1 min at 370 to complete 25 gg of calf-thymus DNA and the indicated concentration of initiation and release of the sigma factor. Modified core enzyme KCL. (10 ,ug) was then added and incubation was continued for 10 min. Control tube with holoehzyme was incubated for 11 min (A); control tube with modified core enzyme was incubated for 10 min molecular weight of 495,000. Modified core and holoenzymes (B). The values are expressed as cpm of [14C]GMP incorporated sediment at 14.5 S and i5.5-16.1 S, respectively. These values into trichloroacetic acid-insoluble material. % Stimulation is represent a molecular weight of 493,000 for modified core en- defined as the difference in the activity of combined enzymes zyme and 587,000 for modified holoenzyme. Compared to their (line 3) and that of normal holoenzyme alone (line 1) compared counterparts in uninfected cells, modified core and holo- to that of core enzyme alone (line 2). enzymes are heavier by about 95,000 and 98,000 daltons, respectively. These results suggest that the increase in molecular weight is localized in the core moiety of the holo- TABLE 3. Sedimentation values and corresponding molecular polymerase. This modification can result from (a) rearrange- weights of normal and modified core and holopolymerases ment of the core component to contain two additional -a subunits, or (b) addition of an entirely new polypeptide chain Increase in to the normal core enzyme, or (c) a modification of a preexist- molecular ing subunit(s) to increase the molecular weight of the com- weight ponent(s). Refer- Molecular over To distinguish between these various possibilities we Enzyme ence S weight reference Increase analyzed the subunit structure of the modified holo- and core sample enzyme value X 10-3 enzyme due to polymerases by sodium dodecyl sulfate-acrylamide-gel Normal electrophoresis along with samples of normal holo- and core holo- polymerases. The gels were stained with Coomassie brilliant enzyme Core 14.4 491 91 Sigma factor blue and scanned at 550 nm to determine the relative inten- Modified sities of the various bands. All samples were judged to be more core than 95% pure, and both modified core and holopolymerases enzyme Core 14.5 493 93 Modification differed from their normal counterparts in only the position Modified and relative amount of the (3' subunit in the #'# region. The (3' core subunit from enzyme Holo 14.5 493 93 Modification the modified RNA polymerase exhibits slower Modified Sigma and electrophoretic mobility* and greater intensity of staining, holo- modifica- indicative of increased molecular weight over normal (3' sub- enzyme Core 15.5 587 187 tion unit (Fig. 4). Comparing the intensity of these bands to the a Modified subunit (39,000 daltons) and assuming a linear relationship holo- between the amount of dye bound and the concentration of enzyme Holo 16.1 591 96 Modification protein present, the observed increased intensity in the (3'O re- gion of the modified core is proportional to 89,000 daltons; the The values represent the average of 2-5 determinations in 5- corresponding value for modified holoenzyme is 79,000 daltons. 20% sucrose velocity gradients made in 10 mM Tris buffer (pH Further evidence for modification in the (' subunit was ob- 7.9)-10 mM MgCl2-0.1 mM EDTA-0.1 mM dithiothreitol-0.5 tained by labeling RNA polymerase during T3 phage infec- M KCl. The gradients were centrifuged for 8 hr at 147,000 X g in an SW 50.1 rotor at 4°. Three-drop fractions were collected and * It is difficult to directly estimate the molecular weight of the fl' assayed for polymerase activity. Catalase position was deter- subunit in modified RNA polymerase by Na dodecyl sulfate- mined by its absorption at 405 nm. Molecular weight calculations acrylamide-gel electrophoresis without the use of marker proteins were based on the values of 12.5 S and 400,000 for core poly- whose molecular weights fall in this region. merase. Downloaded by guest on October 2, 2021 2848 Cell Biology: Dharmgrongartama et al. Proc. Nat. Acad. Sci. USA 70 (1973)

1l 28 - jAI a dye 102 U ( 24[

201

0- 16 x E U) c 12 a ~~~ ~ ~ ~ ~ ~ ~ ~ ~ .k 8

.0 Qa 4 us 7.qw 10 20a 30 40 5u 60 70 80 90 100 Slice Number

FIG. 5. Na dodecyl sulfate-polyacrylamide-gel electrophoresis of labeled RNA polymerases isolated from E. coli B cells infected with either wild-type T3 phage (0) or T3 amH5 (0).

tive. They may bind to phage DNA and interfere with phage- - Direction of Migration specific mRNA synthesis catalyzed by the T3-induced T3 FIG. 4. Na dodecyl sulfate-5% polyacrylamide-gel electro- RNA polymerase. Alternatively, they may continue to syn- phoretogram of ,x' and fi subunits of normal (A) and modified ^ thesize cellular mRNA, thereby decreasing the amount of holopolymerases (B). 20 ,Ag of each polymerase purified on a RNA precursors and consequently protein precursors avail- DNA-cellulose column was analyzed by published methods (7, 8). able to the phage. To achieve high efficiency in this infected system, excess RNA polymerase must be inhibited or in- tion with ['4C]amino acids. RNA polymerase was then pre- activated or both. We have earlier reported on isolation of a cipitated with rabbit antiserum prepared against modified protein from T3-infected cells that markedly inhibits E. coli RNA polymerase. The precipitate was solubilized and ana- RNA polymerase (4). The present results indicate that in lyzed by Na dodecyl sulfate-polyacrylamide-gel electrophore- addition to the production of this inhibitory protein, coli- sis. Fig. 5 shows that almost all the radioactivity in the pre- phage T3 modifies part of the host RNA polymerase after in- cipitate obtained from cells infected with either wild-type fection, resulting in a less catalytically active class of RNA T3 phage or with the gene-1 mutant, T3 amH5, is located polymerase molecules. This decrease in specific activity is an in the ,3' subunit of modified RNA polymerase. Very little inherent property of the purified enzyme due to modification radioactivity appears in the other subunits. A control experi- of the #' subunit, the subunit responsible for DNA binding. ment in which uninfected cells were labeled and subjected Infection of E. coli B cells with the gene-1 amber mutant to the same treatment gave negligible incorporation in all also shows two peaks of enzyme activity on DEAE-cellulose the subunits. T7 infection of E. coli B cells does not result column chromatography, similar to that observed with wild- in any significant incorporation of radioactivity into pre- type T3 phage. Again the late-eluting fraction (0.34 M KCl) cipitated RNA polymerase. These findings, in conjunction can be removed from DNA-cellulose columns only with 0.58 with the increases in molecular weight of modified core and M NaCl. The modified holopolymerase isolated from cells holoenzymes, suggest that host RNA polymerase is probably infected with either wild-type T3 phage or gene-1 mutant modified in the 3' subunit. Thus, in modified RNA poly- has the same sedimentation value in velocity sedimentation merase, the ,#' subunit is heavier than normal g' subunit by studies. Infection of E. coli B cells with either wild-type T3 79,000-89,000 daltons. phage or the gene-1 mutant yielded labeling in the jl' sub- unit of the RNA polymerase (Fig. 5). These findings indicate DISCUSSION that the modification of host RNA polymerase described Like T7, transcription of the T3 is a two-step pro- here may be due to an early gene function. Alternatively, it cess (9-12). Initially the left-hand segment of the phage may well be a late T3 phage function expressed by read- genome (comprising about 20% of the phage genome) is trans- through transcription by host polymerase in the absence of cribed by the host RNA polymerase. This early region con- functional T3-specific RNA polymerase. tains a gene (gene 1) that codes for T3-specific RNA poly- An analogous modification does not seem to occur when a merase responsible for transcription of the remaining 80% closely related T7 phage infects E. coli. This observation of the genome. T3 phage can cause lysis of its host 13 min agrees with that of Schweiger et al. (13), who have reported after infection at 370, and the first progeny phage particle that RNA polymerase purified from that system is normal. is detectable as early as 9 min (9). Such rapid development reflects the extreme efficiency of this system; it is reasonable We thank Drs. R. Hausmann and F. W. Studier for the phage stocks and bacterial strains. This investigation was supported to predict that in infected cells, any host RNA polymerase by grants from the National Science Foundation (GB-23802) and molecules present in excess of the amount required by the the National Institutes of Health (CA-12235). B.D. is supported phage would be potentially dangerous, if not counter-produc- by National Institutes of Health Training Grant GM-255. Downloaded by guest on October 2, 2021 Proc. Nat. Acad. Sci. USA 70 (1973) E. coli RNA Polymerase and T3 Phage Infection 2849

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