Modification of RNA Polymerase after T3 Phage Infection of Escherichia coli B

Abstract

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...

Recognition Mechanisms of DNA-Specific Enzymes

“…The enzyme responsible fo r transcription occurs in several fo rms, including those arising by phage-induced covalent modifications (334)(335)(336). It remains to be established how these various proteins differ in their modes of interaction with DNA.…”

An outer membrane porin protein modulates phage susceptibility in Edwardsiella ictaluri

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