Mutant of Escherichia coli which blocks T7 bacteriophage assembly: Accumulation of short T7 DNA

Yamada, Yoshihiko; Silnutzer, Janet; Nakada, Dai

doi:10.1016/0022-2836(78)90264-4

Cited by 12 publications

(17 citation statements)

References 22 publications

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“…1B, the lysate from T7-infected Y49 cells contained a small amount of phage particles, about 10% of that in T7-infected D10 cells, and a large amount of slow-sedimenting phage head-related particles. This is in agreement with the previous observation that Y49 cells do not produce much of T7 phage in spite of almost normal synthesis of T7-specific macromolecules including DNA (16). In Y49 cells, T7 progeny DNA is not packaged into phage particles.…”

Section: Resultssupporting

confidence: 93%

“…In T7-infected Y49 cells, all T7-specific proteins are sequentially synthesized, and T7 DNA replication proceeds through concatemeric intermediary molecules. However, concatemer DNA molecules are later abnormally cleaved and generate DNA molecules shorter in size than the T7 phage DNA (16). Since the cleavage of concatemeric T7 DNA to unit size is closely related to the process of DNA packaging into the phage heads (8,12,14), the abnormality in cleaving concatemer DNA in T7infected Y49 cells appears to be related to a defect in DNA packaging or to the formation of phage head-related precursor structures requiring an involvement of host function.…”

mentioning

confidence: 99%

“…T7 phage infection. E. coli D10 F-and a mutant strain Y49 (16) were grown in M9 medium containing 0.4% glucose, 50 ug of L-methionine per ml, and 2.5 jig of thiamine per ml at 30°C to a cell density of 3 x 108/ ml and infected with T7 phage at a multiplicity of infection of 10 as described previously (16). For the labeling of proteins with [3S]methionine, the concentration of L-methionine in M9 medium was reduced to 5 ,ug/ml.…”

mentioning

confidence: 99%

“…[35S]methionine-labeled proteins were analyzed by 10% acrylamide-0.1% sodium dodecyl sulfate (SDS) slab gel electrophoresis and radioautographed as described previously (16).…”

mentioning

confidence: 99%

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Accumulation of bacteriophage T7 head-related particles in an Escherichia coli mutant

Yamada¹,

Silnutzer²,

Nakada³

1979

J Virol

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Section: Resultssupporting

confidence: 93%

mentioning

confidence: 99%

mentioning

confidence: 99%

“…[35S]methionine-labeled proteins were analyzed by 10% acrylamide-0.1% sodium dodecyl sulfate (SDS) slab gel electrophoresis and radioautographed as described previously (16).…”

mentioning

confidence: 99%

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Accumulation of bacteriophage T7 head-related particles in an Escherichia coli mutant

Yamada¹,

Silnutzer²,

Nakada³

1979

J Virol

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“…SMBHb likely contacts downstream DNA and forms, together with the downstream jaw domain of ␤Ј, the downstream DNA-binding trough. The downstream jaw is the binding site of T7 Gp2, a protein whose biologically significant function is to lower the stability of host RNAP complexes on viral DNA (8,11,26,29,45; Savalia, Molineux, and Severinov, unpublished). Though we did not test this expressly, it is likely that a negative charge introduced by phosphorylation of Thr 1068 weakens SMBHb interaction with downstream DNA and also destabilizes transcription complexes directly or indirectly (this would explain the lower specific activity of ␤ЈT1068E RNAP).…”

Section: Discussionmentioning

confidence: 99%

Localization of theEscherichia coliRNA Polymerase β′ Subunit Residue Phosphorylated by Bacteriophage T7 Kinase Gp0.7

Severinova

Severinov

2006

J Bacteriol

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During bacteriophage T7 infection, the Escherichia coli RNA polymerase ␤ subunit is phosphorylated by the phage-encoded kinase Gp0.7. Here, we used proteolytic degradation and mutational analysis to localize the phosphorylation site to a single amino acid, Thr 1068 , in the evolutionarily hypervariable segment of ␤. Using a phosphomimetic substitution of Thr 1068 , we show that phosphorylation of ␤ leads to increased -dependent transcription termination, which may help to switch from host to viral RNA polymerase transcription during phage development.Bacteriophage T7 is a prototypic member of a large family of phages that use host RNA polymerase (RNAP) for expression of their early genes but rely on their own, single-subunit RNAP for middle and late viral gene expression. The early T7 genes include, in addition to gene 1 coding for T7 RNAP, several genes whose products participate in host transcription shutoff. The shutoff occurs concurrently with the beginning of transcription of viral genes by T7 RNAP.The products of two viral genes, Gp0.7 and Gp2, are implicated in host transcription shutoff (33)(34)(35)39). Gp0.7 is a serine/threonine protein kinase (31,36). Among the identified targets are components of translation (IF1, IF2, IF3, elongation factor G, and ribosomal proteins S1 and S6 [37,38]) and transcription (RNAP ␤Ј subunit [47]) machineries; RNase III (24), the enzyme responsible for mRNA processing; and RNase E (23), which controls mRNA decay. Several lines of evidence suggest that Gp0.7 plays an important role in bacteriophage development. First, even though the 0.7 gene is dispensable for T7 growth under standard laboratory conditions, it is essential at elevated temperatures or in nutrient-poor media (19), as well as in the presence of certain extrachromosomal genetic elements (16). Second, the protein kinase activity appears to be conserved: it is present in T7-like coliphages (25, 41), as well as in phages that infect Yersinia enterocolitica (32), Kluyvera cryocrescens (15), Klebsiella pneumoniae (D. Savalia, I. J. Molineux, and K. Severinov, unpublished observation), and Caulobacter crescentus (20).Gp2 is a 64-amino-acid-long protein that binds to and abolishes promoter recognition by host RNAP (28). Gene 0.7 becomes essential when Gp2 function is attenuated by a mutation in its binding site in a functionally dispensable domain of RNAP ␤Ј (18), supporting the idea that phosphorylation of ␤Ј by Gp0.7 is functionally relevant and suggesting that Gp2 and Gp0.7 may cooperate with each other during bacteriophage development.Phosphorylation is a common covalent modification of proteins. In eukaryotes, core components of all three nuclear RNAPs are phosphorylated (4, 7). Phosphorylation of the largest subunit, A190, which is homologous to ␤Ј, may regulate the function of yeast (Saccharomyces cerevisiae) RNAP I (14). However, with the exception of the largest RNAP II subunit C-terminal domain phosphorylation, RNAP phosphorylation sites have not been mapped and functional consequences of RNAP phosphorylat...

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Inhibition of Escherichia coli RNA Polymerase by Bacteriophage T7 Gene 2 Protein

Nechaev

Severinov

1999

Journal of Molecular Biology

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Mutant of Escherichia coli which blocks T7 bacteriophage assembly: Accumulation of short T7 DNA

Cited by 12 publications

References 22 publications

Accumulation of bacteriophage T7 head-related particles in an Escherichia coli mutant

Accumulation of bacteriophage T7 head-related particles in an Escherichia coli mutant

Localization of theEscherichia coliRNA Polymerase β′ Subunit Residue Phosphorylated by Bacteriophage T7 Kinase Gp0.7

Inhibition of Escherichia coli RNA Polymerase by Bacteriophage T7 Gene 2 Protein

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