Isolation of a New RNA Polymerase-Binding Protein from Sporulating
            <i>Bacillus subtilis</i>

Greenleaf, Arno L.; Linn, Thomas; Losick, Richard

doi:10.1073/pnas.70.2.490

Cited by 53 publications

(38 citation statements)

References 12 publications

(8 reference statements)

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“…Radioactivity in the precipitates derived from uninfected cells (IS) was located only in the subunits of core RNA polymerase. As previously observed (10) antiserum to core RNA polymerase does not precipitate the a subunit of RNA polymerase efficiently. Radioactivity derived from the SP01-infected cells RNA polymerase was precipitated from one tenth of this partially purified enzyme by the addition of antiserum to RNA polymerase and analyzed by SDS-gel electrophoresis as described in (A) (0, 3H; *, 35S).…”

Section: Methodssupporting

confidence: 52%

“…Solutions were as described by Weber and Osborn (17). The techniques for slicing and solubilizing radioactive gels were as described (10). Double label radioactivity data was corrected for crossover radiation using a computer program written by T. Landers.…”

Section: Methodsmentioning

confidence: 99%

“…Partially purified rabbit antiserum, a gift of T. Linn, was prepared against core RNA polymerase purified from uninfected B. subtilis as previously described (16). Antibody precipitation reactions and solubilization of antibody precipitates for sodium dodecyl sulfate (SDS) gel electrophoresis were carried out as described previously (10).…”

Section: Methodsmentioning

confidence: 99%

“…For example, RNA polymerase isolated from sporulating B. subtilis is associated with two new large polypeptides of 70,000 (10) and 95,000 (11,12) daltons. The 70,000-dalton protein binds to RNA polymerase during precipitation by anti-RNA polymerase antiserum, but it is dissociated from polymerase during phase partitioning (12) or phosphocellulose chromatography (10).…”

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confidence: 99%

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New Phage-SPO1-Induced Polypeptides Associated with Bacillus subtilis RNA Polymerase

Fox

Pero

1974

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

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RNA polymerase was precipitated from extracts of Bacillus subtilis infected with phage SPOI by antiserum prepared against core RNA polymerase. As shown by sodium dodecyl sulfate gel electrophoresis, the precipitates contained at least five new polypeptides not present in uninfected bacteria, in addition to the known subunits of RNA polymerase. The molecular weights of these polypeptides are (1) 85,000; (II) 40,000; (II) 28,000; (lV) 25,000; and (V) 23,000. Four of the polypeptides (I, III, IV, and V) co-purified with RNA polymerase through gel filtration and phosphocellulose chromatography. A pulsechase experiment indicated that all five polypeptides are synthesized de novo after infection. The synthesis of polypeptides II, III, and IV commences almost immediately after infection, while polypeptides I and V first appear several minutes later. A sus mutant blocked early in transcription, susF21 [Fujita, et al. (1971) SP01 is a large virulent phage of Bacillus subtilis containing a double-stranded DNA genome of about 108 daltons (1). After infection, SP01 directs the synthesis of six different classes of phage-specified transcripts in a defined temporal sequence (2). This program of phage RNA synthesis requires the rifampicin-sensitive component of the DNA-dependent RNA polymerase of the host bacteria (3) and is controlled by at least three different regulatory genes of the phage (4). However, the molecular mechanism of this regulation is not yet understood.One possible mechanism for the control of phage transcription is by the direct interaction of regulatory proteins of the phage with host RNA polymerase (5). For instance, phage T4 induces four new small polypeptides that bind to Escherichia coli RNA polymerase (6). Two of these proteins have been shown to be the products of regulatory genes 33 (7) and 55 (D. Ratner, personal communication). Also recent reports indicate that RNA polymerase from B. amyloliquefaciens infected with phage 029 contains a new polypeptide of 30,000 daltons (8), and that RNA polymerase from B. subtilis infected with phage SP82 contains at least one new polypeptide of 16,000 daltons (9). However, it is not yet certain whether either of these polypeptides is the product of a regulatory gene.A rapid procedure for isolating RNA polymerase in association with new subunits is by precipitation with antiserum prepared against core RNA polymerase. This method also permits identification of certain subunits of RNA polymerase which dissociate from RNA polymerase during purification. For example, RNA polymerase isolated from sporulating B. subtilis is associated with two new large polypeptides of 70,000 (10) and 95,000 (11, 12) daltons. The 70,000-dalton protein binds to RNA polymerase during precipitation by anti-RNA polymerase antiserum, but it is dissociated from polymerase during phase partitioning (12) or phosphocellulose chromatography (10).To determine whether phage SP01 induces polypeptides that interact directly with B. subtilis RNA polymerase, we have isolated the enzyme f...

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Section: Methodssupporting

confidence: 52%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

New Phage-SPO1-Induced Polypeptides Associated with Bacillus subtilis RNA Polymerase

Fox

Pero

1974

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

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“…A test of these hypotheses will first require the isolation of the inhibitor from sporulating B. subtili&. Although we favor the view that the inhibitor is a sporulation protein, possibly a new RNA polymerase-binding protein (19), it is not excluded that the inhibitor is a nonprotein component of sporulating bacteria whose synthesis or stability is affected by chloramphenicol.…”

Section: Chloramphenicol Restores a Activitymentioning

confidence: 99%

Chloramphenicol Restores Sigma Factor Activity to Sporulating Bacillus subtilis

Segall

Tjian

Pero

et al. 1974

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

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The a subunit of RNA polymerase from sporulating Bacillus subtilis is markedly inhibited in its ability to direct active transcription of phage 4e DNA in vitro. Treatment of sporulating bacteria with chloramphenicol rapidly restores a activity, suggesting that sporulating cells contain an inhibitor ofa that is physiologically unstable or that becomes unstable after drug treatment. The hypothetical inhibitor is depleted exponentially with an apparent half-life of 11 min at 37°.The onset of sporulation by Bacillus subtilis is associated with a marked decrease in the ability of the a subunit of RNA polymerase to direct active transcription of phage Ye DNA in vitro (1, 2). Although sporulating bacteria contain as much a polypeptide as vegetative bacteria, a from sporulating B. subtilis is functionally inhibited and only weakly associated with RNA polymerase (3). It was previously observed that B. subtilis phage ye is unable to grow in sporulating bacteria (4) and that the time course of decrease in phage burst size during the first hours of spore formation closely parallels the decrease in a activity, as indicated by the reduced ability of RNA polymerase to transcribe he DNA in vitro (5). In contrast, a sporulation-defective mutant of B. subtilis that partially retains a activity supports 4e growth during late stationary phase (6).To test the idea that the failure of he to grow in wild-type sporulating cells is related to the inhibition of a activity (5) (Fig. 1), we found that the rates of total RNA synthesis after infection of vegetative and sporulating bacteria were approximately 0.95 and 0.17 nmol/min per 108 cells, respectively (Table 1). Next we determined the fraction of RNA labeled at 2-4 min after infection that hybridized to denatured he DNA ( Fig. 2A). About 22% of the pulse-labeled RNA from infected vegetative cells and about 11% of the radioactive RNA from infected sporulating bacteria annealed to Oe DNA. From the rates of total RNA synthesis, and correcting for the efficiency of hybridization, we calculate that the rate of Be transcription in sporulat- The RNA extracted from the infected and pulse-labeled cells of the experiments of Fig. 2A and B was hybridized to heavy (H) strand DNA of B. subtilis and to denatured be DNA (Fig. 2) and the % input hybridized at near saturating amounts of DNA was determined. The rates of total cellular RNA synthesis were estimated from the initial slopes of the curves of Fig. iD (the data are the average of two independent experiments).

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Bacillus Subtilis RNA Polymerase and its Modification in Sporulating and Phage‐Infected Bacteria

Losick¹,

Pero²

1976

Advances in Enzymology - And Related Areas of Molecular Biology

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Isolation of a New RNA Polymerase-Binding Protein from Sporulating Bacillus subtilis

Cited by 53 publications

References 12 publications

New Phage-SPO1-Induced Polypeptides Associated with Bacillus subtilis RNA Polymerase

New Phage-SPO1-Induced Polypeptides Associated with Bacillus subtilis RNA Polymerase

Chloramphenicol Restores Sigma Factor Activity to Sporulating Bacillus subtilis

Bacillus Subtilis RNA Polymerase and its Modification in Sporulating and Phage‐Infected Bacteria

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