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Modification of RNA Polymerase After T3 Phage Infection of Escherichia Coli B (DEAE-Cellulose, Phosphocellulose, and DNA-Cellulose Chromatography/H' Subunit)

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Modification of RNA Polymerase After T3 Phage Infection of Escherichia Coli B (DEAE-Cellulose, Phosphocellulose, and DNA-Cellulose Chromatography/H' Subunit) Proc. Nat. Acad. Sci. USA Vol. 70, No. 10, pp. 2845-2849, October 1973 Modification of RNA Polymerase after T3 Phage Infection of Escherichia coli 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 nucleoside triphosphate (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 gene-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 virus 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).
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