An Inhibitory Protein of
            <i>Escherichia coli</i>
            RNA Polymerase in Bacteriophage T3-Infected Cells

Mahadik, Sahebarao P.; Dharmgrongartama, B.; Srinivasan, Prakash

doi:10.1073/pnas.69.1.162

Cited by 22 publications

(8 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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.…”

mentioning

confidence: 72%

“…% Stimulation is defined as the difference in the activity of combined enzymes (line 3) and that of normal holoenzyme alone (line 1) compared to that of core enzyme alone (line 2). 4. Na dodecyl sulfate-5% polyacrylamide-gel electrophoretogram of ,x' and fi subunits of normal (A) and modified holopolymerases (B).…”

Section: Methodsmentioning

confidence: 99%

“…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 M phosphate buffer (pH 7.2) containing 1% Na dodecyl sulfate, 0.5 M mercaptoethanol, and 10% glycerol, by heating in a boiling-water bath for 10 min.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

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

Dharmgrongartama¹,

Mahadik²,

Srinivasan³

1973

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

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...

show abstract

mentioning

confidence: 72%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

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

Dharmgrongartama¹,

Mahadik²,

Srinivasan³

1973

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…However, this does not seem to be the case. In contrast to the T7 repressor, the T3 inhibitor appears to be a late protein, as indicated by the time of induction in vivo (16). It was shown that prior infection with either UVirradiated T3 or with T3 gene-1 mutants-which still could induce the early proteins, except for the gene-1 productdid not reduce significantly phage T4 development.…”

Section: Regulation Of Host Enzymesmentioning

confidence: 99%

“…Recently, an inhibitor of transcription in T3-infected cells was described (16). Since T3 is related to T7, one could imagine that this T3 inhibitor might be closely related to the T7 repressor.…”

Section: Regulation Of Host Enzymesmentioning

confidence: 99%

Negative Control of Protein Synthesis after Infection with Bacteriophage T7

Schweiger

Herrlich

Scherzinger

et al. 1972

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

T7 phage induces two negative control mechanisms of protein synthesis: (a) Host-gene expression is repressed by a " T7 repressor," and (b) The mechanisms by which bacteriophage T7 regulates protein synthesis are only partially known. Soon after infection, a group of phage proteins (early proteins) is synthesized temporarily in a sequential manner. This time sequence is caused by a sequential arrangement of the early genes in an early transcription unit (1). The early transcription unit is transcribed by host RNA polymerase, and corresponds to a total RNA of 2.4 X 108 daltons (2). Within the early region, which is located at one end of the T7 genome, with the promotor close to the end (3), the genes for T7 RNA polymerase (4), ligase, the unknown gene-2 product, endonuclease, and lysozyme are clustered (1). Phage-specific polymerase transcribes the late genes (5). Parallel to these positive controls of phage protein synthesis, host RNA and protein synthesis ceases during phage T7 development. Protein synthesis is necessary for inhibition of host-enzyme synthesis (6). Formation of phage polymerase does not seem to be required for interference with protein synthesis of the host. Production of T7 RNA polymerase, however, is necessary for another negative control: the depression of T7 ligase synthesis (1). The mechanisms of both negative regulations are unknown. Here, we report that for the repression of host-protein synthesis an early protein of T7 is essential. This "repressor" protein is not the product of one of the known early genes. Its gene is located promotor-proximal to the RNA polymerase gene. The "T7 repressor" probably acts on the level of initiation of protein synthesis. The formation of early T7 proteins is depressed by a mechanism that needs newly synthesized T7 RNA polymerase. METHODSProtein Synthesis In Vitro. "Brij-extracts" (7) and "DEAEsystem (8) were described (see Fig. 2).Crude Extracts for Enzyme Assays. For the determination of most enzyme activities in vivo, 5 to 8 X 108 cells were harvested on ice containing 50 A&g/ml of chloramphenicol, and lysed by the Brij-lysozyme method for low concentrations of cells (7). For determination of lysozyme activity, the cells were either sonicated or lysed by freeze-thawing (5 X 108 cells/0

show abstract

Untersuchungen an T3‐Phagen. V. Weitere Charakterisierung von zwei temperatursensitiven Mutanten, ts21 und ts25, in Escherichia coli B und E. coli AB 2500

Scholz

Mann

Hansen

et al. 1973

J of Basic Microbiology

View full text Add to dashboard Cite

Untersuchungen an T3-Phagen V. Weitere Charakterisierung von zwei temperatursensitiven Mutanten, ts21 und ts25, in Escherichia coli B und E. coli AB 2500Zwei temperatursensitive Mutanten des Phagen T3, ts21 und ts25, repriisentative Vertreter zweier Komplementierungsgruppen, wurden in ihrem Verhalten auf E. coli B und E. coli AB2500 niiher charakterisiert. ts21 ist auf beiden Stiimmen bei 41,5 "C nicht zur DNS-Synthese befiihigt, kann Wirts-DNS weitgehend abbauen und beide Wirte lysieren. ts25 ergibt auf E. coli B bei 41,5 O C DNS-Synthese, Lyse sowie einen stark verringerten und verzogerten Wirts-DNS-Abbau, jedoch uberraschenderweise auf mit geringen UV-Dosen bestrahltem E . coli AB2500 keine DNS-Synthese. Bei permissiven Temperaturen verhiilt sich ts25 auf UV-bestrahltem E. coli AB2500 wie T3+. Auf unbestrahltem E. coli AB2500 kann ts25 bei 41,5 "C eine DNS-Synthese induzieren, jedoch bleibt die biologische Komplementierung mit ts21 trotzdem aus.Aus den vorliegenden Daten kann geschlossen werden, daD ts21 wahrscheinlich im Gen fur die DNS-Polymerase mutiert ist. ts25 diirfte seine Mutation in einem der fur den Abbau der Wirts-DNS verantwortlichen Gene tragen. Die Ursachen fur das ungewohnliche Verhalten von ts25 auf E . coli AB2500 sowie fur das Ausbleiben der Komplementierung von ts21 + ts25 auf diesem Wirt sind unbekannt.

show abstract

An Inhibitory Protein of Escherichia coli RNA Polymerase in Bacteriophage T3-Infected Cells

Cited by 22 publications

References 18 publications

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

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

Negative Control of Protein Synthesis after Infection with Bacteriophage T7

Untersuchungen an T3‐Phagen. V. Weitere Charakterisierung von zwei temperatursensitiven Mutanten, ts21 und ts25, in Escherichia coli B und E. coli AB 2500

Contact Info

Product

Resources

About