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At least 19 recordsLinked to original sources
PubMed1988

Initiation of DNA replication at cloned origins of bacteriophage T7.

Bacteriophage T7 DNA replication is initiated at a site 15% of the distance from the genetic left end of the chromosome. This primary origin contains two tandem T7 RNA polymerase promoters (phi 1.1A and phi 1.1B) followed by an A + T-rich region. When the primary origin region is deleted replication initiates at secondary origins. We have analyzed the ability of plasmids containing cloned fragments of T7 to replicate after infection of Escherichia coli with bacteriophage T7. All cloned T7 fragments that support plasmid replication contain a T7 promoter but a T7 promoter alone is not sufficient for replication. Replication of plasmids containing the primary origin is dependent on T7 DNA polymerase and gene 4 protein (helicase/primase) and a portion of the A + T-rich region. The other T7 fragments that support plasmid replication after T7 infection are promoter regions phi OR, phi 13 and phi 6.5 (secondary origins). When both the primary and secondary origins are present simultaneously on compatible plasmids, replication of each is temporally regulated. Such regulation may play a role during T7 DNA replication.

Base Sequence↗
PubMed1968

Production of bacteriophage T7.

Bacteriophage T7 was grown with Escherichia coli B as the host organism in 3- and 20-liter vessels. Under the best growth conditions devised, the yields of T7 in the culture lysates averaged 1.33 x 10(12) and 0.95 x 10(12) plaque-forming units per ml, respectively, compared with the best previously reported yields of 10(11) to 3 x 10(11) plaque-forming units per ml in 1-liter batches grown in the presence of air, or double this in similar batches grown in the presence of oxygen. The bacteriophage was purified by a simple method which gave average yields of 143 mg/liter and 131 mg/liter from the 3- and 20-liter batches, respectively. The efficiency of plating of the final material ranged from 18 to 42%. The purified bacteriophage is a convenient source of monodisperse deoxyribonucleic acid, the molecular weight of which is about 25 x 10(6).

Bacteriolysis↗
PubMed1984

In vitro template activity of 0.3 mRNA from wild type and initiation mutants of bacteriophage T7.

Bacteriophage T7 0.3 mRNA synthesised and processed in vitro has been purified starting from the DNA of T7+ as well as from that of two initiation mutants of T7 (CR17 with a U----C transition in the initiation codon and CR35b whose potential Shine and Dalgarno (S-D) interaction is interrupted by a G----A transition). These mRNAs were used as templates to direct the binding of fMet-tRNA and the synthesis of 0.3 protein in both E. coli and wheat germ cell-free systems. The initiation codon mutant displayed approximately 50% inhibition of fMet-tRNA binding and 0.3 protein synthesis in both systems. The S-D sequence mutant, on the other hand, was found to be less affected than the initiation triplet mutant (20%-40% inhibition) in both fMet-tRNA binding and template activity in the E. coli system. In the wheat germ system, which does not make use of the S-D interaction, however, this mutant displayed normal template activity suggesting that the inhibition obtained in the E. coli system, albeit slight, is due to the impairment of the S-D interaction and not to an alteration of the mRNA secondary or tertiary structure caused by the base substitution.

Cell-Free System↗
PubMed1992

Interactions of gene 2.5 protein and DNA polymerase of bacteriophage T7.

Bacteriophage T7 gene 2.5 protein has been shown to interact with T7 DNA polymerase (the complex of T7 gene 5 protein and Escherichia coli thioredoxin) by affinity chromatography and fluorescence emission anisotropy. T7 DNA polymerase binds specifically to a resin coupled to gene 2.5 protein and elutes from the resin when the ionic strength of the buffer is raised to 250 mM NaCl. In contrast, T7 gene 5 protein alone binds more weakly to gene 2.5 protein, eluting when the ionic strength of the buffer is 50 mM NaCl. Thioredoxin does not bind to gene 2.5 protein. Steady-state fluorescence emission anisotropy gives a dissociation constant of 1.1 +/- 0.2 microM for the complex of gene 2.5 protein and T7 DNA polymerase, with a ratio of gene 2.5 protein to T7 DNA polymerase in the complex of 1:1. Nanosecond emission anisotropic analysis suggests that the complex contains one monomer each of gene 2.5 protein, gene 5 protein, and thioredoxin. The ability of T7 gene 2.5 protein to stimulate the activity and processivity of T7 DNA polymerase is compared with the ability of three other single-stranded DNA-binding proteins: E. coli single-stranded DNA-binding protein, T4 gene 32 protein, and E. coli recA protein. All except E. coli recA protein stimulate the activity and processivity of T7 DNA polymerase; E. coli recA protein inhibits these activities.

Base Sequence↗
PubMed1989

Benzo[a]pyrene-DNA adducts inhibit translocation by the gene 4 protein of bacteriophage T7.

Bacteriophage T7 gene 4 protein is an essential component of the T7 DNA replication system, acting as both a primase and a helicase. The gene 4 protein has been shown to translocate along single-stranded DNA in the 5'----3' direction, using an energy source for this movement the hydrolysis of nucleoside 5'-triphosphates, preferably dTTP. Thus, measuring the rate and extent of dTTP hydrolysis provides a means to directly measure translocation. We have determined that the hydrolysis of dTTP by the gene 4 protein is strongly inhibited by the presence of benzo[a]pyrene (B[a]P) adducts on the DNA. Time course experiments on adduct-containing DNA show that after an initial burst of hydrolysis, which parallels what is observed on unmodified DNA, further hydrolysis abruptly ceases. Addition of excess unmodified DNA does not restore the hydrolysis activity. These data suggest that the gene 4 protein is blocked and sequestered on the DNA at the site of the adduct. This was confirmed by experiments in which gene 4 protein preferentially protected the radiolabeled adduct-containing DNA but not randomly labeled M13 DNA. The gene 4 protein bound to the B[a]P-modified DNA was isolated, and the complex was found only to contain dTTP. These results have been used to formulate a model for gene 4 protein translocation in which we speculate that the power stroke for unidirectional movement along the single-stranded DNA is the displacement of dTDP by dTTP. Finally, we observe a constant ratio of DNA synthesis to dTTP hydrolysis regardless of the number of B[a]P adducts in the template suggesting that a significant portion of the inhibition of DNA synthesis is a direct consequence of the inhibition of gene 4 translocation.

Benzo(a)pyrene↗
PubMed1987

Escherichia coli thioredoxin confers processivity on the DNA polymerase activity of the gene 5 protein of bacteriophage T7.

Bacteriophage T7 gene 5 protein has been purified to apparent homogeneity from cells overexpressing its gene several hundred-fold. Gene 5 protein is a DNA polymerase with low processivity; it dissociates from the primer-template after catalyzing the incorporation of 1-50 nucleotides, depending on the salt concentration. Escherichia coli thioredoxin, a host protein that is tightly associated with the gene 5 protein in phage-infected cells, is not required for this activity. Thioredoxin acts as an accessory protein to bestow processivity on the polymerizing reaction; DNA synthesis catalyzed by the gene 5 protein-thioredoxin complex on a single-stranded DNA template can polymerize thousands of nucleotides without dissociation. Conditions that increase the stability of secondary structures in the template (i.e., low temperature or high ionic strength) decrease the processivity. E. coli single-stranded DNA-binding protein stimulates both the rate of elongation and the processivity of the gene 5 protein-thioredoxin complex.

DNA-Directed DNA Polymerase↗
PubMed1977

Intracellular organization of bacteriophage T7 DNA: analysis of parenteral bacteriophage T7 DNA-membrane and DNA-protein complexes.

After infection of Escherichia coli with bacteriophage T7, the parenteral DNA forms a stable association with host cell membranes. The DNA-membrane complex isolated in cesium chloride gradients is free of host DNA and the bulk of T7 RNA. The complex purified through two cesium chloride gradients contains a reproducible set of proteins which are enriched in polypeptides having molecular weights of 54,000, 34,000, and 32,000. All proteins present in the complex are derived from host membranes. Treatment of the complex with Bruij-58 removes 95% of the membrane lipid and selectively releases certain protein components. The Brij-treated complex has an S value of about 1,000 and the sedimentation rate of this material is not altered by treatment with Pronase or RNase.

Bacterial Proteins↗
PubMed1975

Bacteriophage T7 deoxyribonucleic acid replication invitro. Bacteriophage T7 DNA polymerase: an an emzyme composed of phage- and host-specific subunits.

The DNA polymerase induced after infection of Escherichia coli by phage T7 has been purified 500-fold to near homogeneity as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified enzyme complements extracts of cells infected with a T7 gene 5 mutant to permit cell-free replication of duplex T7 DNA. In contrast, purified T4 DNA polymerase or E. coli DNA polymerase I is unable to do so, thus suggesting a specific requirement for the T7 enzyme in the replication of the viral DNA. E. coli TsnC protein is present in purified T7 DNA polymerase in one-to-one stoichiometry with T7 gene 5 protein, and can be isolated in homogeneous form from heat-denatured enzyme by chromatography on DEAE-cellulose. The inactive form of T7 gene 5 protein that accumulates in tsnC hosts has been partially purified. When partially purified gene 5 protein is mixed with purified TsnC protein, DNA polymerase activity is restored, and formation of a one-to-one complex between the two proteins occurs. These results indicate that the functional form ofT7 DNA polymerase is a complex composed of phage- and host-specified subunits.

Bacterial Proteins↗
PubMed1997

Mechanism of inhibition of bacteriophage T7 RNA polymerase by T7 lysozyme.

Bacteriophage T7 lysozyme is known to inhibit transcription by T7 RNA polymerase. Lysozyme present before initiation inhibited the synthesis of long RNA chains but did not inhibit elongation when added shortly after chains were initiated. A combination of gel-shift and transcription assays showed that lysozyme and polymerase form a 1:1 complex that binds promoter DNA and makes abortive transcripts, indicating that lysozyme has little effect on the early steps of transcription. Extension of stalled transcription complexes suggested that a transcribing polymerase becomes resistant to lysozyme inhibition after synthesis of an RNA chain as short as 15 nucleotides. It seems likely that bound lysozyme prevents an initiating polymerase from converting to an elongation complex. This conversion is thought to involve both a conformational change in the polymerase and the binding of nascent RNA. Gel-shift experiments indicated that lysozyme does not interfere with the binding of RNA, so it probably prevents a necessary conformational change in the polymerase. Lysozyme also increased pausing or termination at two sites in lambda DNA and at a site near the right end of the concatemer junction of T7 DNA. If pausing at these sites involves a reversal from the elongation to the initiation conformation, lysozyme may increase pausing or termination by "locking in" the initiation conformation. The arrest of transcription complexes near promoters and near the right end of the concatemer junction almost certainly must relate to lysozyme's ability to stimulate replication, maturation and packaging of T7 DNA during T7 infection.

Bacteriophage T7↗
PubMed1983

A recombinant DNA plasmid which inhibits bacteriophage T7 reproduction in escherichia coli.

Escherichia coli plasmids carrying a DNA restriction fragment corresponding to the extreme right end of the T7 bacteriophage genome render cells incompetent to support reproduction of wild-type T7. Processing of intracellular concatemeric phage DNA and cell lysis are perturbed by one such plasmid, pRS148. The results are discussed with reference to the possibility that the right end of the T7 genome encodes a lysis-related function.

DNA Replication↗
PubMed2000

Nucleoside analogue substitutions in the trinucleotide DNA template recognition sequence 3'-(CTG)-5' and their effects on the activity of bacteriophage T7 primase.

Bacteriophage T7 primase catalyzes the synthesis of the oligoribonucleotides pppACC(C/A) and pppACAC from the single-stranded DNA template sites 3'-d[CTGG(G/T)]-5' and 3'-(CTGTG)-5', respectively. The 3'-terminal deoxycytidine residue is conserved but noncoding. A series of nucleoside analogues have been prepared and incorporated into the conserved 3'-d(CTG)-5' site, and the effects of these analogue templates on T7 primase activity have been examined. The nucleosides employed include a novel pyrimidine derivative, 2-amino-5-(beta-2-deoxy-D-erythro-pentofuranosyl)pyridine (d2APy), whose synthesis is described. Template sites containing d2APy in place of the cryptic dC support oligoribonucleotide synthesis whereas those containing 3-deaza-2'-deoxycytidine (dc(3)C) and 5-methyl-6-oxo-2'-deoxycytidine (dm(5ox)C) substitutions do not, suggesting that the N3 nitrogen of cytidine is used for a critical interaction by the enzyme. Recognition sites containing 4-amino-1-(beta-2-deoxy-D-erythro-pentofuranosyl)-5-methyl-2,6[1H, 3H]-pyrimidione (dm(3)2P) or 2'-deoxyuridine (dU) substitutions for dT support oligoribonucleotide synthesis whereas those containing 5-methyl-4-pyrimidinone 2'-deoxyriboside (d(2H)T) substitutions do not, suggesting the importance of Watson-Crick interactions at this template residue. Template sites containing 7-deaza-2'-deoxyguanosine (dc(7)G) or 2'-deoxyinosine (dI) in place of dG support oligoribonucleotide synthesis. The reduced extent to which dc(7)G is successful within the template suggests a primase-DNA interaction. Inhibition studies suggest that the primase enzyme binds "null" substrates but cannot initiate RNA synthesis.

Bacteriophage T7↗
PubMed2026

Replication of DNA Containing Trinucleotide Repeats by the Bacteriophage T7 Replisome.

Trinucleotide repeats in the human genome are implicated in various neurodegenerative diseases. The tendency of these repetitive DNA sequences to form non-B DNA structures can cause abnormal replication, leading to genomic instability. This instability contributes to disease progression, though the underlying mechanisms are not fully understood. We investigated the replication of DNA containing CAG and CTG trinucleotide repeats using individual components of the T7 bacteriophage replication machinery, as well as the complete replisome. Our results show that repeats in linear single-stranded DNA (ssDNA) inhibit the activity of T7 DNA polymerase and ssDNA-binding proteins, with a more pronounced effect observed in CTG repeats compared to CAG repeats. Direct unwinding assays showed that the T7 gene 4 helicase unwound forked substrates containing CAG or CTG repeats at least as efficiently as random-sequence substrates; however, the displaced repeat strands were recovered predominantly as compact, structured species rather than as unstructured single-stranded DNA, providing direct evidence that secondary structure forms immediately upon unwinding. Minicircle templates containing CTG repeats exhibited robust DNA synthesis on both the leading and lagging strands, though synthesis was not enhanced by the T7 gene 2.5 ssDNA-binding protein. The lagging strand products generated from the CTG repeat minicircle were significantly longer than those from random sequence templates, and their lengths were not extended by the presence of T7 gene 2.5 protein. When the repeated sequences were incorporated into the T7 phage genome, heterogeneity was observed downstream of the repeats, depending on their length. We propose that aberrant extension occurs predominantly in the lagging strand, driven by dynamic interactions between the repeated sequences and the DNA replisome. This study may provide a foundation for understanding the mechanisms underlying the extension or deletion of repetitive genomic regions.

DNA repeats↗
PubMed1989

Sequence-dependent termination of bacteriophage T7 transcription in vitro by DNA-binding drugs.

An in vitro T7 bacteriophage transcription system has been utilized in which the RNA was initiated to a specific length (defined by the absence of the appropriate nucleoside triphosphate). When the DNA-RNA-RNA polymerase ternary complex was exposed to nonsaturating levels of DNA-binding ligands (i.e., a small fractional occupancy at each site), and the RNA transcript then allowed to elongate in the presence of all four nucleoside triphosphates, there was a synchronous increase of RNA lengths up to sites occupied by ligands. A unique characteristic is that bacteriophage transcription was completely terminated at every ligand site, in contrast to bacterial RNA polymerases where "read-through" past drug sites occurs and results merely in a delay of transcription at each site due primarily to dissociation of drug from the DNA. Similar termination of transcription at each drug site was observed with T3 and SP6 RNA polymerases. The termination at drug sites in the bacteriophage system results in RNA of specific lengths which define the location of ligand sites, and the RNA concentration provides a measure of relative ligand occupancy at that site. Termination of transcription was observed with four drugs with relatively long DNA residence times (half-life greater than or equal to 300 s at 20 degrees C for nogalamycin, actinomycin, mithramycin, and echinomycin) but to a lesser extent with drugs of intermediate residence times [a bis(thiadaunomycin) and an acridine-tripyrrole, with half-lives of 230 and 7 s, respectively, at 20 degrees C]

Anti-Bacterial Agents↗
PubMed1992

Transcription by an immobilized RNA polymerase from bacteriophage T7 and the topology of transcription.

It is often assumed that a polymerase moves along the template as it synthesizes RNA. However, a polymerase that tracks along a helical strand will generate a transcript that is entwined about the template. No such interlocking results if the polymerase is immobile and the template moves past it. Therefore we investigated whether immobilization inhibits the RNA polymerase of T7 bacteriophage using a hybrid protein, in which the polymerase is connected through a peptide linker to an immobilizing domain, which in turn was attached through an antibody to protein A covalently linked to plastic beads. Polymerase could be released by cleaving the linker with a protease, factor Xa. Comparison of the activity of the bound and free enzymes showed that immobilization reduced the rate of initiation about fivefold. However, when re-initiation was eliminated by removing excess template, immobilization was found to have little effect on the rate of elongation. Perhaps the untwining problem is sidestepped in vivo by immobilizing the polymerase.

Base Sequence↗
PubMed1982

Escherichia coli mutant which restricts T7 bacteriophage has an altered RNA polymerase.

We have previously described an Escherichia coli K-12 mutant, Y49, which restricts the growth of bacteriophage T7 and causes the accumulation of short DNA molecules and head-related particles during infection. We now show that the basis for these effects is the inability of the T7 gene 2 product to inactivate the Y49 RNA polymerase during infection, similar to what has been shown by DeWyngaert and Hinkle (J. Biol. Chem. 254:11247--11253, 1979) for the BR3 and tsnB strains of E. coli.

DNA-Directed RNA Polymerases↗