Cloning and generation of a genetic map of bacteriophage N4 DNA

Malone, C.; Spellman, S.; Hyman, David A.; Rothman-Denes, Lucia B.

doi:10.1016/0042-6822(88)90472-2

Cited by 15 publications

(13 citation statements)

References 17 publications

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“…In work with bacterial strains containing expression vectors, IPTG (0.4 mM) and ampicillin (0.2 mg/ml) were added to the top agar at the time of plating. Phage mutations were considered rescued when the wild-type PFU/total PFU ratio (efficiency of rescue) obtained after plating on a nonsuppressing host was 100-to 1,000-fold higher than the ratio obtained by using mutant phage stocks grown in a host containing a control plasmid (35).…”

Section: Bacterial Strains and Phages Escherichia Coli K-12 Strains mentioning

confidence: 99%

“…After addition of 2 mM isopropyl-␤-D-thiogalactopyranoside (IPTG) to induce expression of T7 RNAP and subsequent transcription of the N4 genes, followed by incubation for 30 min at 37°C, 200 g of rifampin/ml was added to inhibit host transcription. After incubation for 90 min at 37°C, 50-l samples were labeled with 5 l of Tran 35 S-label (Ͼ1,000 Ci/mmol; ICN Radiochemicals, Irvine, Calif.) for 10 min at 37°C. Cells were collected by centrifugation and processed as described previously (14).…”

Section: Bacterial Strains and Phages Escherichia Coli K-12 Strains mentioning

confidence: 99%

“…DNA fragments generated by HpaI digestion of N4 genomic DNA and cloned into pBR322 (35) were transferred to M13mp18 or M13mp19. Viral ssDNA was isolated and sequenced by the chain termination technique using Sequenase T7 DNA polymerase (version 2.0) and reagents purchased from United States Biochemical Corp. (USB).…”

Section: Bacterial Strains and Phages Escherichia Coli K-12 Strains mentioning

confidence: 99%

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N4 RNA Polymerase II, a Heterodimeric RNA Polymerase with Homology to the Single-Subunit Family of RNA Polymerases

Willis¹,

Kazmierczak²,

Carter

et al. 2002

J Bacteriol

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Bacteriophage N4 middle genes are transcribed by a phage-coded, heterodimeric, rifampin-resistant RNA polymerase, N4 RNA polymerase II (N4 RNAPII). Sequencing and transcriptional analysis revealed that the genes encoding the two subunits comprising N4 RNAPII are translated from a common transcript initiating at the N4 early promoter Pe3. These genes code for proteins of 269 and 404 amino acid residues with sequence similarity to the single-subunit, phage-like RNA polymerases. The genes encoding the N4 RNAPII subunits, as well as a synthetic construct encoding a fusion polypeptide, have been cloned and expressed. Both the individually expressed subunits and the fusion polypeptide reconstitute functional enzymes in vivo and in vitro.Sequence and structural analysis of DNA-dependent RNA polymerases (RNAPs) conclusively supports the existence of two enzyme families. One family consists of the large, evolutionarily related, multisubunit polymerases of eubacteria, archaea, and the nuclear and chloroplast polymerases of eukaryotes (reference 56 and references therein). Single-subunit enzymes with sequence homology to the T3/T7 phage polymerases belong to the second family of DNA-dependent RNAPs (38).Transcription of coliphage N4 middle mRNAs requires the activities of three early proteins: p17, p7, and p4 (53,70,72). Two of these gene products, p7 (30 kDa) and p4 (40 kDa), are soluble, have been purified to homogeneity, and constitute a heterodimeric, rifampin-resistant RNAP, N4 RNAPII (71). However, this heterodimer does not transcribe promoter-containing, double-stranded DNAs (dsDNA) and utilizes singlestranded DNAs (ssDNA) with low efficiency and no specificity. At least one additional phage-coded protein, p17, is essential in vivo for N4 middle RNA synthesis but is not sufficient in vitro for utilization of N4 middle promoters (1, 70; R. H. Carter, unpublished data). Recent in vitro characterization of p17 indicates that p17 is a ssDNA binding protein that recruits N4 RNAPII to ssDNA templates (R. H. Carter and A. Demidenko, unpublished data).In the present study, the genes encoding the p7 and p4 subunits of N4 RNAPII were sequenced and cloned. Both genes are transcribed from the same phage early promoter. The two subunits display sequence similarity to separate, nonoverlapping regions of single-subunit, T7-like RNAPs, suggesting that gene fusion or gene splitting events have occurred during the evolution of this class of polymerases. An N4 fusion polypeptide was generated that is active in vitro and complements phages carrying mutations in either of the two subunits when expressed in vivo. Ϫ m K12 ϩ recA1) was used in the construction of expression vectors. Protein expression was carried out in either BL21(DE3) (62) or W3350(DE3)pLysS (11). Phage were grown as described previously (53). MATERIALS AND METHODS Bacterial strains and phages. Escherichia coliPreparation and manipulation of DNAs. N4 DNA was prepared according to the work of VanderLaan et al. (64). DNA amplification reaction mixtures contained 1 g of tem...

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Section: Bacterial Strains and Phages Escherichia Coli K-12 Strains mentioning

confidence: 99%

Section: Bacterial Strains and Phages Escherichia Coli K-12 Strains mentioning

confidence: 99%

Section: Bacterial Strains and Phages Escherichia Coli K-12 Strains mentioning

confidence: 99%

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N4 RNA Polymerase II, a Heterodimeric RNA Polymerase with Homology to the Single-Subunit Family of RNA Polymerases

Willis¹,

Kazmierczak²,

Carter

et al. 2002

J Bacteriol

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“…An HpaI restriction digest library of the N4 genome was used to define complementation groups. N4am229 was rescued by the HpaI E fragment, which encodes the N-terminal 136 residues of gp64, the entirety of gp65, and the C-terminal 136 residues of gp66 (20). PCR amplification of this region of the N4am229 genome, followed by sequence analysis, revealed that the amber mutation was located in Orf65, resulting from a G-to-T transversion at nucleotide 1297 and a consequent mutation of a glutamine codon (aa 433) to a stop codon (GAG 3 TAG).…”

Section: Isolation Of 3 H-labeled N4gp65mentioning

confidence: 99%

The Tail Sheath of Bacteriophage N4 Interacts with the Escherichia coli Receptor

McPartland

Rothman-Denes

2009

J Bacteriol

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Unlike other characterized phages, the lytic coliphage N4 must inject the 360-kDa virion RNA polymerase (vRNAP), in addition to its 72-kbp genome, into the host for successful infection. The process of adsorption to the host sets up and elicits the necessary conformational changes in the virion to allow genome and vRNAP injection. Infection of suppressor and nonsuppressor strains, Escherichia coli W3350 supF and E. coli W3350, with a mutant N4 isolate (N4am229) harboring an amber mutation in Orf65 yielded virions containing (N4gp65 ؉ ) and lacking (N4gp65 ؊ ) gp65, respectively. N4gp65 ؉ but not N4gp65 ؊ phage was able to adsorb to the host. Recombinant gp65 with a hexahistidine tag at the N terminus or hexahistidine and c-myc tags at the C terminus was able to complement N4gp65؊ virions in vivo and in vitro. Immunogold detection of gp65 in vivo complemented virions revealed its localization at the N4 tail. Finally, we show both in vitro and in vivo that gp65 interacts with the previously determined N4 outer membrane receptor, NfrA.

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“…pBRK, a plasmid containing the N4 HpaIK fragment (Malone et al 1988), was isolated from W3350 (F-, Cou R, gal, lac). CLM 363 is an M13 mp9 derivative that carries the PM103 BamHI fragment containing the P1 promoter at its BamHI site…”

Section: Dna Templatesmentioning

confidence: 99%

Escherichia coli single-stranded DNA-binding protein is a supercoiled template-dependent transcriptional activator of N4 virion RNA polymerase.

Markiewicz¹,

Malone²,

Chase³

et al. 1992

Genes Dev.

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Coliphage N4 is a double-stranded DNA virus that requires the sequential activity of three different RNA polymerases during infection. The N4 virion RNA polymerase, which is carried in the virion and is injected with the DNA at the start of infection, is responsible for the synthesis of N4 early RNAs. In vitro, the virion RNA polymerase can transcribe double-stranded N4 DNA accurately and efficiently but only when the DNA is denatured. We have shown previously that the activity of DNA gyrase is required for in vivo early N4 transcription. We report here that Escherichia coil single-stranded DNA-binding protein (SSB) is also required for N4 early transcription. In vitro, linear or relaxed templates cannot be activated by SSB; however, supercoiled template and SSB allow the virion polymerase to recognize its promoters on duplex DNA and activate transcription. The effects of supercoiling are limited to transcript initiation and are not required for transcript elongation. The activation is specific for SSB; no other single-stranded DNA-binding proteins can substitute. Therefore, SSB is one of a small number of proteins that function to stimulate both replication and transcription. The basis for the specificity of SSB, the mechanism of transcriptional activation by SSB and template supercoiling, and their role in the N4 transcriptional program during development are discussed.[Key Words: Coliphage N4; RNA polymerase; single-stranded DNA-binding protein; transcriptional activation; template supercoiling] Bacteriophage N4 is a double-stranded DNA virus specific for Escherichia coli K 12 strains. Transcription of its genome is regulated through the sequential activity of three distinct RNA polymerases (for review, see Kiino and Rothman-Denes 1988). Early N4 transcripts are synthesized by a rifampicin-resistant, N4-coded RNA polymerase, which is carried within the virion and injected into the cell at the start of infection (Falco et al. 1977). N4 middle transcripts are synthesized by a second N4-coded RNA polymerase (N4 RNA polymerase II) (Zehring and Rothman-Denes 1983; . Late in infection, the N4-coded, single-stranded DNA-binding protein directs the host RNA polymerase to transcribe the N4 structural genes (Zivin et al. 1981; N. Baek, M. Choi and L. Rothman-Denes, in prep.) Purified virion RNA polymerase shows peculiar template specificity. It is unable to use native duplex genomic N4 DNA as a template (Falco et al. 1978(Falco et al. , 1980 polymerases, it transcribes denatured or single-stranded, promoter-containing templates accurately and efficiently (Haynes and Rothman-Denes 1985; Glucksmann et al. 1992). This result suggests that the structure of N4 DNA must be altered upon entry into the cell to allow transcription by the virion RNA polymerase. It is likely that the N4 virion RNA polymerase utilizes a supercoiled template because active host DNA gyrase is required for N4 early RNA synthesis (Falco et al. 1978). However, in vitro, supercoiled, promoter-containing templates do not support virion RNA polymeras...

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Cloning and generation of a genetic map of bacteriophage N4 DNA

Cited by 15 publications

References 17 publications

N4 RNA Polymerase II, a Heterodimeric RNA Polymerase with Homology to the Single-Subunit Family of RNA Polymerases

N4 RNA Polymerase II, a Heterodimeric RNA Polymerase with Homology to the Single-Subunit Family of RNA Polymerases

The Tail Sheath of Bacteriophage N4 Interacts with the Escherichia coli Receptor

Escherichia coli single-stranded DNA-binding protein is a supercoiled template-dependent transcriptional activator of N4 virion RNA polymerase.

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