Conversion of Bacillus subtilis RNA polymerase activity in vitro by a protein induced by phage SP01.

Duffy, John; Petrusek, R.; Geiduschek, E. Peter

doi:10.1073/pnas.72.6.2366

Cited by 38 publications

(13 citation statements)

References 15 publications

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“…Like all RNA polymerases from prokaryotic origin B. subtilis enzyme has the subunit composition tII 3 I 4 2, 6C . However, some reports suggest that RNA polymerase may be part of a more complex transcriptive machinery (6,7) and that prokaryotic cells may contain RNA polymerase activities heterogenous in terms of different template specificities (8)(9)(10) and subunit composition (11,12). In order to investigate these possibilities we have purified the RNA polymerase from B. subtilis by a method that is based on mild lysis of cells, low speed centrifugation, gel filtration, DEAE-Sephadex chromatography Chemicals.…”

Section: Introductionmentioning

confidence: 99%

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RNA polymerase from Bacillus subtilis: isolation of core and holo enzyme by DNA-cellulose chromatography

et al. 1977

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A new procedure for the purification of B. subtilis RNA polymerase, based on mild lysis of cells, low speed centrifugation, gel filtration, DEAE-Sephadex chromatography and affinity chromatography on DNA-cellulose, yields three forms of enzyme referred here as enzyme A, B and C. As revealed by SDS gel electrophoresis, enzyme A has the subunit structure of core polymerase plus some small polypeptides. Its catalytic properties are similar to those of core polymerase. Enzyme B has the composition of core polymerase. Both enzymes A and B can be stimulated by the addition of 6' factor. Enzyme C has the holo-enzyme composition. The pattern of sensitivity of the three forms of enzyme towards KC1 are very different: enzymes A and B, even at low concentration of salt,are inhibited with all the DNA templates tested, whereas enzyme C shows a pattern of stimulation specific for each DNA tested. The transcripts of the three enzymes on phage SPP1 DNA template have been analyzed by hybridization to the separated strands. Only enzyme C selectively transcribed the H strands.

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Section: Introductionmentioning

confidence: 99%

“…The DNA-dependent RNA polymerase from Bacillus subtilis has been purified by a variety of methods (1)(2)(3)(4)(5)(6)(7) . Like all RNA polymerases from prokaryotic origin B. subtilis enzyme has the subunit composition tII 3 I 4 2, 6C .…”

Section: Introductionmentioning

confidence: 99%

RNA polymerase from Bacillus subtilis: isolation of core and holo enzyme by DNA-cellulose chromatography

et al. 1977

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“…A dramatic example is the reprogramming of promoter specificity of Bacillus subtilis RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) after infection with phages such as SP01 or SP82 in which a viral protein replaces the normal B. subtilis a' subunit (1)(2)(3). This type of regulatory mechanism has long been considered an attractive possibility in normal cells (4); however, although many "altered" forms of RNA polymerase have been reported (refs.…”

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confidence: 99%

Heterogeneity of RNA polymerase in Bacillus subtilis: evidence for an additional sigma factor in vegetative cells.

Wiggs¹,

Gilman²,

Chamberlin³

1981

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

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Preparations ofBacillus subtilis RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) from vegetatively growing cells contain small amounts of an activity (B. subtilis RNA polymerase holoenzyme H) that shows a unique promoter specificity with T7 bacteriophage DNA as compared with the normal B. subtilis holoenzyme (holoenzyme I) A large variety of molecular mechanisms are known by which organisms regulate transcription during growth and development. A dramatic example is the reprogramming of promoter specificity of Bacillus subtilis RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) after infection with phages such as SP01 or SP82 in which a viral protein replaces the normal B. subtilis a' subunit (1-3). This type of regulatory mechanism has long been considered an attractive possibility in normal cells (4); however, although many "altered" forms of RNA polymerase have been reported (refs. 5-11, see refs. 12-14 for reviews), it is only quite recently that bacterial RNA polymerases have been detected that clearly express novel promoter specificities (15,16 MATERIALS AND METHODSExperimental procedures and materials have been described previously (16) except where noted. Growth ofB. subtilis W168 in early exponential phase and preparation of fraction 5 RNA polymerase proceeded as before (16) except that: (i) precipitation of RNA polymerase with ammonium sulfate employed 0.4 g/ml after Polymin fractionation and 0.42 g/ml after DNA cellulose chromatography; (ii) the precipitate from the first ammonium sulfate fractionation was desalted by chromatography on Bio-Gel P-10 (Bio-Rad) in a buffer containing 10 mM Tris-HCl (pH 8), 1 mM EDTA, 1 mM dithiothreitol, 10% (vol/ vol) glycerol, and 50 mM NaCl; (iii) RNA polymerase was eluted from the DNA cellulose column with a 4 column volume gradient (0.05-1.0 M NaCl).B. subtilis core RNA polymerase was prepared by chromatography of peak fractions of RNA polymerase holoenzyme I from heparin agarose (fractions HA 116-132) on phosphocellulose (21) followed by rechromatography on poly(rC)-cellulose (22) to remove a contaminating polypeptide of Mr 92,000. RNA polymerase from the phosphocellulose column (1.5 mg) in 10 mM Tris-HCl (pH 8)/1 mM EDTA/10 mM 2-mercaptoethanol/ 10 mM MgCl2/10% (vol/vol) glycerol/0.02 M NaCl was passed through a 2-ml poly(rC)-cellulose column. Flow-through fractions were pooled with those eluted with 0.1 M NaCl and were concentrated by dialysis against 50% (vol/vol) 2762The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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“…In addition, the positive control probably operates at initiation rather than antitermination (Fig. 2) (21).…”

Section: Discussionmentioning

confidence: 99%

Synthesis of specific functional messenger RNA in vitro by phage-SP01-modified RNA polymerase of Bacillus subtilis.

Swanton¹,

Smith²,

Shub³

1975

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

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RNA polymerase (nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) was purified from rifampicin-resistant Bacillus subtifis, from both uninfected cells and cells infected with bacteriophage SPOl. The enzyme from infected cells lacked all traces of the sigma subunit, contained several polypeptides absent from the enzyme made in uninfected cells, and had an altered template specificity in a transcription assay. A cell-free protein synthesizing system from Escherichia coli, when poisoned with rifampicin, was completely dependent on addition of either of these RNA polymerase preparations for DNA-dependent protein synthesis. Under these conditions, the SPOI-modified RNA polymerase preferentially stimulated the synthesis of functional mRNA for the phage enzyme dCMP deaminase (deoxycytidylate aminohydrolase, EC 3.5.4.12), whereas unmodified B. subtilis RNA polymerase could stimulate synthesis of this mRNA in small quantity and only after prolonged incubation. This mRNA belongs to a class of phage transcripts (m) which cannot be transcribed in vivo in the absence of phage-specific protein synthesis.Transcription of the genome of bacteriophage SP01 has been extensively studied by competitive RNA-DNA hybridization (1). Six classes of transcripts have been described, which are independently controlled with respect to the turning on or turning off of their synthesis. Two of these (e and em) are synthesized after infection without detectable lag, and are subsequently repressed if phage protein synthesis is allowed to occur. Three additional classes of RNA (m, mll and m21) are made independently of phage DNA replication, but require prior phage protein synthesis. In particular, the protein product of cistron 28 is required for m and m1l expression, and the protein products of cistrons 33 and 34 are required for M21 RNA synthesis (2,3). After phage DNA synthesis begins, another population of transcripts (1) is made.The existence of three pre-replicative classes of phagespecific transcripts (m, m1l, and M21), whose synthesis is under phage genetic control, suggested the possibility that mature phage DNA extracted from purified virus might be an appropriate template for a new RNA polymerase (nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) of the correct specificity for these classes. Since it had been shown that the rifampicin sensitivity of RNA synthesis throughout SP01 infection was determined by the sensitivity of the host's RNA polymerase (4) it was logical to expect that the new specificity for middle RNA synthesis should reside in the host's RNA polymerase, which had been modified in some way. Duffy and Geiduschek (5) demonstrated that the expected in vitro selectivity for phage middle transcripts does, indeed, reside in an enzyme which contains the host core and other peptides which are made after phage infection.Competitive RNA-DNA hybridization is a very powerful tool which can be used to determine the relative amount of specific nucleotide sequences in a population of in vitro synth...

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Conversion of Bacillus subtilis RNA polymerase activity in vitro by a protein induced by phage SP01.

Cited by 38 publications

References 15 publications

RNA polymerase from Bacillus subtilis: isolation of core and holo enzyme by DNA-cellulose chromatography

RNA polymerase from Bacillus subtilis: isolation of core and holo enzyme by DNA-cellulose chromatography

Heterogeneity of RNA polymerase in Bacillus subtilis: evidence for an additional sigma factor in vegetative cells.

Synthesis of specific functional messenger RNA in vitro by phage-SP01-modified RNA polymerase of Bacillus subtilis.

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