Physical map of coliphage N4 DNA

Zivin, Robert A.; Malone, C.; Rothman-Denes, Lucia B.

doi:10.1016/0042-6822(80)90378-5

Cited by 23 publications

(3 citation statements)

References 12 publications

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“…To elucidate the template requirements for specific vRNAP transcription, sites of transcription initiation were mapped both in vivo and in vitro to the nucleotide level, revealing three sites of vRNAP initiation (P1, P2, and P3) within the leftmost 10% of the genome [ 19 , 20 , 21 ]. Sequences spanning −17 to +1 (relative to the transcription start site at +1) are conserved across all three promoters and include a GC-rich heptamer centered at −11 flanked by inverted repeats [ 21 ].…”

Section: N4 Transcriptional Architecturementioning

confidence: 99%

Structural and Biochemical Investigation of Bacteriophage N4-Encoded RNA Polymerases

Lenneman

Rothman-Denes

2015

Biomolecules

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Bacteriophage N4 regulates the temporal expression of its genome through the activity of three distinct RNA polymerases (RNAP). Expression of the early genes is carried out by a phage-encoded, virion-encapsidated RNAP (vRNAP) that is injected into the host at the onset of infection and transcribes the early genes. These encode the components of new transcriptional machinery (N4 RNAPII and cofactors) responsible for the synthesis of middle RNAs. Both N4 RNAPs belong to the T7-like "single-subunit" family of polymerases. Herein, we describe their mechanisms of promoter recognition, regulation, and roles in the phage life cycle.

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Section: N4 Transcriptional Architecturementioning

confidence: 99%

Structural and Biochemical Investigation of Bacteriophage N4-Encoded RNA Polymerases

Lenneman

Rothman-Denes

2015

Biomolecules

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“…Here, we report the purification, ATPase activity and crystallization of the putative large terminase protein from bacteriophage N4, a lytic phage that infects the Escherichia coli K12 strain (Molina et al, 1965). Phage N4 possesses a linear dsDNA genome of 72 kbp and encodes 72 proteins (Zivin et al, 1980;Ohmori et al, 1988). A remarkable feature of phage N4 is that it packages 3-5 copies of the virion-encoded RNA polymerase (vRNAP) along with its genome, which is utilized for transcription of the early viral genes during infection (Rothman-Denes & Schito, 1974;Falco et al, 1977).…”

Section: Introductionmentioning

confidence: 99%

Bacteriophage N4 large terminase: expression, purification and X-ray crystallographic analysis

Wangchuk

Prakash

Bhaumik

et al. 2018

Acta Crystallogr F Struct Biol Commun

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Genome packaging is a critical step in the assembly of dsDNA bacteriophages and is carried out by a powerful molecular motor known as the large terminase. To date, wild-type structures of only two large terminase proteins are available, and more structural information is needed to understand the genome-packaging mechanism. Towards this goal, the large and small terminase proteins from bacteriophage N4, which infects the Escherichia coli K12 strain, have been cloned, expressed and purified. The purified putative large terminase protein hydrolyzes ATP, and this is enhanced in the presence of the small terminase. The large terminase protein was crystallized using the sitting-drop vapour-diffusion method and the crystal diffracted to 2.8 Å resolution using a home X-ray source. Analysis of the X-ray diffraction data showed that the crystal belonged to space group P222, with unit-cell parameters a = 53.7, b = 93.6, c = 124.9 Å, α = β = γ = 90°. The crystal had a solvent content of 50.2% and contained one molecule in the asymmetric unit.

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“…Coliphage N4, a lytic podovirus with a 72 kb linear double-stranded dsDNA genome (Zivin et al, 1980), has a characteristically long infectious cycle with lysis occurring more than 3 h after infection (Schito, 1974). N4-infected cells continue to grow in length and diameter throughout the period of infection while cell division is blocked, thus increasing several-fold in size and producing over 3000 phage per infected cell (Schito, 1974).…”

Section: Introductionmentioning

confidence: 99%

A phage‐encoded inhibitor of Escherichia coli DNA replication targets the DNA polymerase clamp loader

Yano

Rothman-Denes²

2011

Molecular Microbiology

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SummaryColiphage N4 infection leads to shut-off of host DNA replication without inhibition of host transcription or translation. We report the identification and characterization of gp8, the N4 gene product responsible for this phenotype. N4 gp8 is an Escherichia coli bacteriostatic inhibitor that colocalizes with the E. coli replisome in a replication-dependent manner. Gp8 was purified and observed to cross-link to complexes containing the replicative DNA polymerase, DNAP III, in vivo. Purified gp8 inhibits DNA polymerization by DNA polymerase III holoenzyme in vitro by interfering with polymerase processivity. Gp8 specifically inhibits the clamp-loading activity of DNAP III by targeting the delta subunit of the DNAP III clamp loader; E. coli mutations conferring gp8 resistance were identified in the holA gene, encoding delta. Delta and gp8 interact in vitro; no interaction was detected between gp8 inactive mutants and wild-type delta or between delta gp8-resistant mutants and wild-type gp8. Therefore, this work identifies the DNAP III clamp loader as a new target for inhibition of bacterial growth. Finally, we show that gp8 is not essential in N4 development under laboratory conditions, but its activity contributes to phage yield.

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Physical map of coliphage N4 DNA

Cited by 23 publications

References 12 publications

Structural and Biochemical Investigation of Bacteriophage N4-Encoded RNA Polymerases

Structural and Biochemical Investigation of Bacteriophage N4-Encoded RNA Polymerases

Bacteriophage N4 large terminase: expression, purification and X-ray crystallographic analysis

A phage‐encoded inhibitor of Escherichia coli DNA replication targets the DNA polymerase clamp loader

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