Initiation by RNA polymerase on UV or X-ray damaged T7 DNA

Summers, William C.

doi:10.1016/0006-291x(70)91080-6

Cited by 39 publications

(7 citation statements)

References 11 publications

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“…Phage were further purified on a CsCl step gradient followed by equilibrium banding in CsCl. Template DNA was extracted with sodium dodecyl sulfate (NaDodSO4) at 650 (9).…”

Section: Methodsmentioning

confidence: 99%

Highly asymmetric transcription by RNA polymerase containing phage-SP01-induced polypeptides and a new host protein.

Pero¹,

Nelson²,

Fox³

1975

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

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An RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) has been purified from phage-SPOl-infected Bacillus subtilis that copies RNA almost exclusively from the heavy strand of native SPOl DNA, the DNA strand from which "middle" and "late" classes of RNA are copied in vivo. Hybridization-competition established that this RNA polymerase, termed enzyme A, preferentially synthesizes middle RNA in vitro. Enzyme A contains $', , a, and two newly identified host polypeptides, a (21,500 daltons) and co (11,000 daltons). All of these polypeptides are associated with highly purified RNA polymerase from uninfected bacteria.In addition, enzyme A contains phage-induced subunits of 26,000, 24,000, and 13,500 daltons. Enzyme A lacks a-polypeptide, and strand-selective transcription by this enzyme is resistant to anti-a antibody. A reconstitution experiment strongly suggests that the host 5 protein is required in addition to a phage-induced subunit(s) (or an unidentified phage-induced modification) for strand-selective transcription of SPOl middle genes in vitro.The Bacillus subtilis DNA phage SP01 directs the synthesis of six different classes of phage-specified transcripts in a temporally defined sequence (1). Two classes of early RNA (e and em) appear immediately after infection and are copied from both the heavy (H) and light (L) strands of SP01 DNA. This transcription is not inhibited by chloramphenicol and presumably does not require phage-specified protein synthesis. About 4-5 min after infection two classes of middle transcripts (m and mil) are first synthesized largely from strand H (1). This gene transcription requires the protein product of regulatory gene 28 (2, 3). Finally, late RNA transcripts (m21 and 1) do not appear until 8-14 min into the lytic cycle and are transcribed almost exclusively from strand H. Mutations in regulatory genes 33 and 34 block the synthesis of late RNA (1-3).RNA polymerase (nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) isolated from SP01-infected cells is associated with several new phage-induced polypeptides (4). It seemed possible that at least some of these proteins could be regulatory elements that direct the transcription of middle and late genes. In this paper, we report on the purification of an RNA polymerase from infected cells containing phage-induced subunits and a newly identified host polypeptide of 21,500 daltons. This enzyme (enzyme A) selectively transcribes middle genes from the H strand of SP01 DNA, whereas, RNA polymerase from uninfected cells transcribes early genes from both strands of SP01 DNA. As will be discussed, enzyme A is apparently different from other RNA polymerases isolated from phage-infected B. subtilis that have been reported to synthesize middle RNA in titro (5-7). METHODSPreparation of SPOl DNA. Wild-type SP01 (obtained from D. Shub) was grown and partially purified as described (8) except that phage were initially precipitated with polyethylene glycol. Phage were further purified on a CsCl step grad...

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“…Phage were further purified on a CsCl step gradient followed by equilibrium banding in CsCl. Template DNA was extracted with sodium dodecyl sulfate (NaDodSO4) at 650 (9).…”

Section: Methodsmentioning

confidence: 99%

Highly asymmetric transcription by RNA polymerase containing phage-SP01-induced polypeptides and a new host protein.

Pero¹,

Nelson²,

Fox³

1975

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

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“…Introduction of single-strand breaks (86,204) or otlaer lesions (213,214) into has relatively little effect on the selectivity of RNA synthesis by RNA polymerase holoenzyme, although these lesions serve as tight binding sites for the enzyme. This suggests that tr subunit may constrain the enzyme to carry out the initiation reaction only at a given nucleotide sequence.…”

Section: Conditions Affecting Selectivity Of In Vitro Transcriptionmentioning

confidence: 99%

The Selectivity of Transcription

Chamberlin¹

1974

Annu. Rev. Biochem.

529

213

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“…Par contre, en l'absence de rifampicine, des cassures monocatenaires dans le DNA induisent des sites d'initiation non specifiques [19,20]. E n consequence la qualit6 de RNA synth6tisBe L partir de telles matrices peut Btre plus grande que pour une matrice temoin qui ne portent gubre de cassures monocat6-naires.…”

Section: R61e Tventuel De La Dtpurination D a M La Perte D'aetivitt Ounclassified

“…coli resistantes aux rayons ultraviolets reparent non seulement les dommages Crees par les irradiations, mais encore, lcs dommages Crees par les moutardes bifonctionelles [27-291. Chessin et Summers [20] montrent que le DNA irradib par I'ultraviolet est une mauvaise matrice pour la synthbse du RNA. Toutefois l'asymetric de la transcription est conservee.…”

Section: Influence Possible Des Ponts Intracatdnairesunclassified

Action de la moutarde azotée sur le DNA utilisé comme matrice pour la synthèse du RNA

Degré-Couve

Mamet‐Bratley

1973

European Journal of Biochemistry

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The goal of this study is to elucidate the mechanism by which nitrogen mustard, a difunctional alkylating agent, inhibits DNA template activity for RNA synthesis. This inhibition could be due not only monofunctional alkylations, known to hinder RNA synthesis, but also to interand intrastrand bridges formed by difunctional alkylation. DNA from bacteriophage T, and Escherichia coli RNA polymerase provide a well-characterized system for RNA synthesis in vitro. Only 20°/, of the T, genome is transcribed by E . coli RNA polymerase and hence only those alkylations falling in this region could influence RNA synthesis.Three types of experiments were carried out. T, DNA was alkylated by treatment with varying concentrations of nitrogen mustard. This alkylated DNA was used as template for RNA synthesis; the extent of interstrand crosslinking in the DNA was compared with its loss of template activity. Interstrand crosslinks were gradually eliminated from alkylated T, DNA by incubation, under sterile conditions, for variable times at 37 "C; these DNAs were then used as templates for RNA synthesis in vitro; the antibiotic, rifampicin, was included in the synthesis medium to eliminate non-specific chain initiation. T, DNA was alkylated with radioactive nitrogen mustard to calculate the extent of alkylation a t the dose which reduces activity to 3701, of the control.Template activity decreases with increasing doses of nitrogen mustard ; however, the fraction of DNA molecules remaining without interstrand crosslinks decreases much more rapidly. Since only 20°/, of T, DNA is transcribed by E. coli RNA polymerase, we examined the relation between the fraction of molecules bearing one interstrand crosslink in this region and the inhibition of RNA synthesis. The inhibition is significantly greater ; interstrand orosslinks could, a t the most, explain only one third of the activity loss.As interstrand crosslinks disappear from alkylated T, DNA, its template activity, in the presence of rifampicin, decreases. A significant but smaller loss of activity (39°/0 after 72 h of incubation) is observed in the non-alkylated control DNA. When RNA synthesis is carried out in the absence of rifampicin, the activity losses of the control and of the alkylated sample are entirely comparable. Had interstrand crosslinks been responsible for the initial loss of activity after alkylation, their disappearance should have caused a reactivation ; had they been without influence, activity would have been expected to remain constant. We tentatively interpret our result to indicate that other phenomena, i.e. depurination and single strand breakage, which occur during the incubation of alkylated DNA, are responsible for the loss of activity; the possible effect of interstrand crosslinks may thus be masked.At the concentration of nitrogen mustard which reduces DNA template activity to 37O/, of the control, T, DNA is alkylated to the extent of 90 alkyl groups per molecule. This dose is about 9 times less than that necessary with methylation or ethylation of T,...

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Initiation by RNA polymerase on UV or X-ray damaged T7 DNA

Cited by 39 publications

References 11 publications

Highly asymmetric transcription by RNA polymerase containing phage-SP01-induced polypeptides and a new host protein.

Highly asymmetric transcription by RNA polymerase containing phage-SP01-induced polypeptides and a new host protein.

The Selectivity of Transcription

Action de la moutarde azotée sur le DNA utilisé comme matrice pour la synthèse du RNA

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