The expression of bacteriophage T5-specific RNA and protein in infected cells is temporally separated into three classes: class I (preearly), class II (early), and class III (late). By immunoprecipitation techniques we have shown that T5 infection of cells leads to the synthesis of one class I polypeptide (11,000 daltons) and two class II polypeptides (90,000 and 15,000 daltons) capable of binding to the RNA polymerase of the host Escherichia coli cell. One of the class II polypeptides (90,000 daltons) is the product of gene C2, which is an essential gene product required for the initiation of class III RNA synthesis. The colicinogenic factor, ColIb, is a plasmid which prevents the normal synthesis of class II and the III bacteriophage T5-specific RNA in infected colicinogenic (ColIb+) cells. In T5-infected colicinogenic cells, only the T5 class I polypeptide is found associated with the RNA polymerase. Mutants of T5, designated T5h minus, are capable of growth on both noncolicinogenic and ColIb+ hosts. Extracts of T5h minus infected ColIb+ cells were shown to lack a small class I polypeptide (12,000 daltons) as compared to T5-infected cells. The h minus mutation, however, has no effect on the levels of the class I T5 polypeptide of similar molecular weight which is bound to the RNA polymerase. One effect of the h minus mutation is to enhance the quantities of the two class II polypeptides bound to the enzyme.
A partially purified protein isolated from bacteriophage T3-infected cells of Escherichia coli B markedly inhibits the activity of E. coli RNA polymerase, slightly inhibits the activity of purified T3 polymerase, and does not inhibit the activity of either core polymerase or the ribosome-bound T3 polymerase. DEAE-cellulose column (2.2 X 20 cm), which was washed with 150 ml of buffer A; the column was eluted with a gradient from 0 to 0.4 M KCl in buffer A. 5-ml fractions were collected, and the fractions containing RNA polymerase activity (0.23-0.3 M KCl) were pooled and diluted 1:1 with buffer C (identical to buffer A, except for 50 mM Tris HCl), and passed through a phosphocellulose column equilibrated with buffer C containing 0.1 M KCl. The inhibitory activity was eluted with the unadsorbed material. This eluate was adjusted to 10 mM MgCl2 and placed on a second DEAEcellulose column (1.8 X 20 cm). The column was eluted with a gradient from 0 to 0.4 M KCl in buffer A containing 10 mM MgCl2. The fractions (0.25-0.3 M KCl) that contained the inhibitory activity were pooled and precipitated with (NH4)2SO4.
E. coli B cells infected with T3 phage contain a modified host RNA polymerase in addition to the normal RNA polymerase found in uninfected cells. The modified RNA polymerase behaves differently in its elution properties from the normal enzyme on DEAE-cellulose, phosphocellulose, and DNA-cellulose column chromatography. The modified enzyme also differs from the normal polymerase in some of its enzymatic parameters. The specific activity of the modified RNA polymerase is markedly lower (i.e., 1/4) than that of the The mechanisms by which an invading virulent virus can arrest the macromolecular synthesis of its host and initiate synthesis of phage-specific proteins are being studied in several phage-infected systems (1-3). We have previously reported the isolation of an inhibitory protein of Escherichia coli RNA polymerase from E. coli B cells infected with T3 phage, which may be responsible for the arrest of host RNA synthesis (4). During that study we observed that the total activity of host RNA polymerase from T3-infected cells freed from the inhibitory protein and nucleases was considerably lower than the activity of RNA polymerase isolated from uninfected cells. We present evidence here that suggests that this decrease in activity is due to a structural modification of the host RNA polymerase, which is accompanied by various alterations in its enzymatic properties. METHODSPreparation of Polymerases from Normal and Infected Cells. RNA polymerase holoenzyme of E. coli B was prepared by the method of Burgess by ammonium sulfate fractionation and DEAE-cellulose chromatography (5). The enzyme was further purified by chromatography on DNA-cellulose columns (6), or by high-salt and low-salt glycerol gradient centrifugation (5) and then by DNA-cellulose column chromatography. Core polymerase was prepared from the holoenzyme by chromatography on phosphocellulose (5).The modified RNA polymerase was isolated from E. coli B cells infected at 300 with T3 phage at a multiplicity of 5-10 as described (4). 10 min after infection, the cells were rapidly chilled to 40 and harvested by centrifugation.RNA Polymerase Assay. The reaction mixture for RNA synthesis contained in 0.25 ml: 40 mM Tris * HC1 buffer (pH 7.9), 10 Na Dodecyl Sulfate-Polyacrylamide-Gel Electrophoresis of Labeled Polymerases. E. coli B was grown to a cell density of 5 X 108 cells per ml at 370 and then shifted to 300. After 15 min the culture was divided into four equal portions of 20 ml each. One culture was used as a control, and the other three cultures received wild-type T3 phage or T3 amH5 (an amber mutant in gene 1) or T7 phage. 2 min later, 100 uCi of "IClabeled reconstituted protein hydrolysate (Schwarz mixture, Schwarz BioResearch) was added to each flask. After 8 min of incorporation, the cells were rapidly chilled and harvested by centrifugation. For polymerase isolation, each sample was mixed with 1 g of unlabeled E. coli B cells before the cell-free extract was subjected to (NH4)2SO4 fractionation. All solutions contained phenylmethylsu...
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