In vitro transcription of a late class of phage SP01 genes

Pero, Janice; Tjian, Robert; Nelson, Jane; Losick, Richard

doi:10.1038/257248a0

Cited by 38 publications

(18 citation statements)

References 16 publications

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“…In addition to its effect on core RNA polymerase (Fig.3), 6 protein purified in this manner was effective in promoting asymmetric transcription by phage-SPO1 -modified RNA polymerase and has been routinely used in our recent studies of SPOl 'middle' and 'late' gene transcription in vitro [8,9]. We [7] have previously shown that 6 protein can also be separated from (T factor by chromatography on poly(C)-cellulose. However, a dis- advantage of this earlier procedure was that it led to inactivation of cr factor.…”

Section: Discussionmentioning

confidence: 99%

Purification and Comparative Properties of the Delta and Sigma Subunits of RNA Polymerase from Bacillus subtilis

Tjian

Losick

Pero

et al. 1977

European Journal of Biochemistry

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Bacillus subtilis delta protein is a 21 500-Mr polypeptide that can be isolated in association with RNA polymerase holoenzyme from uninfected bacteria and with modified forms of RNA polymerase from cells infected with phage SPOl [Pero, J., Nelson, J. and Fox, T. (1975) Proc. Nut1 Acad. Sci. U.S.A. 72, 15891. Although no function has been assigned to 6 protein in uninfected cells, this host polypeptide enhances the specificity of transcription by phage-modified forms of RNA polymerase that contain SPOI-coded regulatory subunits. This report describes the purification of 6 and B proteins from uninfected B. subtilis and examines the comparative effects of these polypeptides on transcription by core RNA polymerase. Purified B polypeptide was found to stimulate the transcription of phage DNA while having little effect on RNA synthesis with the synthetic DNA poly(dA-dT) as template. In contrast, purified 6 protein markedly depressed the transcription of poly(dA-dT) while having little effect on enzyme activity with phage DNA as template. The inhibitory effect of 6 protein on poly(dA-dT) transcription was strongly dependent on the presence of KCl in the RNA synthesis reaction mixture.DNA-dependent RNA polymerase holoenzyme from a variety of procaryotic sources exhibits the general structure ~' B~Z B [1,2]. The p', p and a subunits constitute a minimal or core enzyme that is sufficient for the polymerization of ribonucleoside triphosphates into RNA. Although not required for RNA synthesis, the o subunit is thought to promote initiation at promoter sites on DNA and to suppress 'incorrect' initiation at non-specific sites [3,4]. Purified RNA polymerase also frequently contains a small polypeptide termed o for which no specific function in transcription has yet been assigned. Other bacterial proteins that appear to interact with RNA polymerase have been considered in several recent reviews [l -51.Recently this laboratory has described a novel Bacillus subtilis protein termed delta ( M I 21 500) that can be isolated in association with highly purified RNA polymerase holoenzyme from uninfected bacteria and with modified forms of RNA polymerase from bacteria infected with phage SPOl [6]. A function for 6 protein in uninfected cells is not known at present. However, recent experiments indicate that 6 protein acts to enhance selective transcription of the temporally defined 'middle' and 'late' genes of phage SPOl by forms of RNA polymerase that contain SPO1-coded regulatory subunits and that lack o Enzyme. DNA-dependent RNA polymerase or nucleosidetriphosphate : RNA nucleotidyltransferase (EC 2.7.7.6).factor [5-81. Selective transcription of the 'early' genes of SPO1, in contrast, is relatively unaffected by 6 protein but is strongly dependent on G factor [9].Here we describe the isolation of 6 and B proteins in pure form from RNA polymerase holoenzyme of uninfected B. subtilis. We further compare the effects of these two proteins on transcription by core RNA polymerase and provide a simple new functional assay for 6 polypept...

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Section: Discussionmentioning

confidence: 99%

Purification and Comparative Properties of the Delta and Sigma Subunits of RNA Polymerase from Bacillus subtilis

Tjian

Losick

Pero

et al. 1977

European Journal of Biochemistry

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“…hr and then treated as described (14). Preparation of RNA Pulse-Labeled In Vivo and the Purification of Unlabeled RNAs.…”

Section: Methodsmentioning

confidence: 99%

“…One milliliter of cells (2.4 X 108) grown in 121A medium (8) was labeled with 100 MCi of [3H]-uridine (28 Ci/mol) for 2 min at the indicated times after infection (multiplicity of infection = 5). The cells were rapidly chilled, brought to 0.01 M in sodium azide, and RNA was extracted as described previously (14). Unlabeled RNA was purified (14) from cells grown in 121 A medium, infected with SP01 (multiplicity of infection = 5-10), chilled at the indicated times after infection by pouring cells over frozen 121 medium containing sodium azide, and harvested by centrifugation.…”

Section: Methodsmentioning

confidence: 99%

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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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“…Our results strongly suggest that phage "early" and "middle" genes are transcribed from distinct promoters and that the RNA polymerase containing the gene 28 protein binds to sites that are located at or near promoters for SPOI "middle" genes. The specificity of gene transcription by RNA polymerase in Bacillus subtilis is modified by infection with bacteriophage SPOI (1)(2)(3)(4). The unmodified RNA polymerase holoenzyme of the host bacteria specifically copies only "early" genes on the phage genome (1,3).…”

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Promoter recognition by phage SP01-modified RNA polymerase.

Talkington¹,

Pero²

1978

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

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A modified form of Bacillus subtilis RNA polymerase containing a phage SPOI-coded regulatory protein (the gene 28 product) selectively transcribes "middle" genes of the phage genome in vitro. In this paper, we identify a subset of restriction endonuclease fragments of SPOl DNA that promote specific transcription by the phage-modified polymerase. In the absence of nucleoside triphosphates, RNA polymerase containing the gene 28 protein selectively binds to these DNA fragments thereby forming stable binary complexes that can be isolated on nitrocellulose filters. In contrast, unmodified RNA polymerase containing sigma factor selectively binds to and transcribes a subset of phage DNA fragments that contain "early" sequences and that are in large part distinct from the fragments recognized by the phage-modified transcriptase. Our results strongly suggest that phage "early" and "middle" genes are transcribed from distinct promoters and that the RNA polymerase containing the gene 28 protein binds to sites that are located at or near promoters for SPOI "middle" genes.

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In vitro transcription of a late class of phage SP01 genes

Cited by 38 publications

References 16 publications

Purification and Comparative Properties of the Delta and Sigma Subunits of RNA Polymerase from Bacillus subtilis

Purification and Comparative Properties of the Delta and Sigma Subunits of RNA Polymerase from Bacillus subtilis

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

Promoter recognition by phage SP01-modified RNA polymerase.

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