A DNA-Binding Protein Induced by Bacteriophage T7

A DNA-binding protein has been purified from Escherichia coli infected with bacteriophage T7 by DNA-cellulose chromatography. The protein is absent in uninfected cells. The purified protein has a molecular weight of 31,000 and binds strongly and preferentially to single-stranded DNA. In vitro studies show that this protein can stimulate the rate of polymerization catalyzed by the T7-induced DNA polymerase 10-15 times under conditions where the polymerase is unable to effectively use a single-stranded template. The degree of stimulation is dependent upon the ratio of binding protein to DNA template and is independent of polymerase concentration.The observed stimulation is specific for the T7 DNA polymerase in that addition of the protein to reactions catalyzed by E. coli DNA polymerases I, II, or III or T4 DNA polymerase is without effect.     

Protein Kinase Induction in Escherichia coli by Bacteriophage T7

After bacteriophage T7 infection, a protein kinase (EC 2.7.1.37; ATP:protein phosphotransferase) activity can be demonstrated in E. coli in vivo by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Cell-free extracts catalyzed the transfer of the terminal phosphoryl group of [(gamma)-(32)P]ATP to endogenous protein acceptor or to added histone. The bond between phosphate and protein shows the characteristics of serine phosphate: it is stable in 1 N HCl (100 degrees ) and cleaved by 1 N KOH (37 degrees ) and by alkaline phosphatase treatment. Moreover, after partial acid hydrolysis, radiophosphate migrates with marker O-phosphoserine on polyethyleneimine-cellulose thin-layer chromatograms. Enzyme activity in uninfected cells is negligible. Ultraviolet irradiation of the phage genome prevents the appearance of the protein kinase; irradiation of the host genome does not. The enzyme activity occurs 4 min after infection and its gene maps in the early region (promoter proximal to gene 1). Ribosomal proteins are phosphorylated in vivo and are substrates in vitro. Enzyme activity in vitro is not changed by addition of cyclic AMP or cyclic GMP.     

Eight Transfer RNAs Induced by Infection of Escherichia coli with Bacteriophage T4

Bacteriophage T4 induces the synthesis of eight transfer RNAs upon infection of E. coli. The tRNAs are easily detected and resolved into pure species by polyacrylamide gel electrophoresis of RNA labeled with ³²P after T4 infection. Two-dimensional fingerprints of RNase T₁ products derived from individual gel bands give patterns characteristic of single tRNAs. Furthermore, the T₁ digest of each gel band has a single oligonucleotide that contains the minor nucleotides Tp and Ψp, a characteristic feature of all known tRNAs.     

Bacteriophage T7 DNA Replication: A Linear Replicating Intermediate

The T7 chromosome in the first round of replication is a Y-shaped DNA rod. Thus, it differs from previously observed bacterial and viral replicating chromosomes that are circular.     

Packaging and Maturation of DNA of Bacteriophage T7 In Vitro

We have developed an in vitro complementation assay to demonstrate packaging and maturation of DNA of phage T7. Cells of Escherichia coli B infected with an appropriate T7 amber mutant are concentrated 200-fold and lysed by freezing and thawing. Two extracts from cells infected with different amber mutants are mixed and incubated at 30 degrees . Positive complementation results in a 100-fold increase in phage titer.Using this assay we have demonstrated the packaging of phage DNA from an extract that contains no phage heads (gene 9(-), 10(-)), within head structures present in an extract that contains no phage DNA (gene 5(-)). We have also demonstrated an activity in extracts that contain no phage DNA or heads (gene 5(-), 9(-), 10(-)), which complements gene 19(-)-infected cells. We have proven that this activity is due to the gene-19 product by showing that the activity is temperature-sensitive if the extract is made from cells infected with a mutant having a temperature-sensitive mutation in gene 19. This assay should be useful in elucidating the mechanism of packaging and maturation of DNA of phage T7.     

Initiation and Reinitiation of DNA Synthesis during Replication of Bacteriophage T7

In its first round of replication, the T7 chromosome follows a simple pattern, as viewed in the electron microscope. The iniation of DNA synthesis occurs about 17% from the genetic left end of the viral DNA rod. Bidirectional DNA synthesis from this origin then generates a replicating intermediate that we call an "eye form." In the eye form, when synthesis in the leftward direction reaches the left end of the viral chromosome, the molecule is converted into a Y-shaped replicating rod. The remaining growing point continues synthesis rightward, until presumably it runs off the right end of the DNA rod, thus terminating replication.Numerous T7 chromosomes were found in which a second round of replication had begun before the first round had finished. Analysis of these reinitiated DNA molecules showed that the second round of replication, like the first, began 17% from the end of the chromosome and involved bidirectional DNA synthesis.     

Coordinated DNA Replication by the Bacteriophage T4 Replisome

The T4 bacteriophage encodes eight proteins, which are sufficient to carry out coordinated leading and lagging strand DNA synthesis. These purified proteins have been used to reconstitute DNA synthesis in vitro and are a well-characterized model system. Recent work on the T4 replisome has yielded more detailed insight into the dynamics and coordination of proteins at the replication fork. Since the leading and lagging strands are synthesized in opposite directions, coordination of DNA synthesis as well as priming and unwinding is accomplished by several protein complexes. These protein complexes serve to link catalytic activities and physically tether proteins to the replication fork. Essential to both leading and lagging strand synthesis is the formation of a holoenzyme complex composed of the polymerase and a processivity clamp. The two holoenzymes form a dimer allowing the lagging strand polymerase to be retained within the replisome after completion of each Okazaki fragment. The helicase and primase also form a complex known as the primosome, which unwinds the duplex DNA while also synthesizing primers on the lagging strand. Future studies will likely focus on defining the orientations and architecture of protein complexes at the replication fork.     

Regions of Single-Stranded DNA in the Growing Points of Replicating Bacteriophage T7 Chromosomes

In partially replicated T7 chromosomes, the points where parental strands are separating and new DNA is being synthesized can be seen in the electron microscope to contain regions of single-stranded template DNA. The single-stranded regions are located on only one of the two daughter arms of the replicating chromosome. Inman and Schnös observed such single-stranded regions in 50% of the growing points of replicating lambda DNA, and, as reported in this paper, we find them in about 85% of the growing points of T7 DNA. Both studies support the conclusion that DNA synthesis involves the direct elongation of one daughter strand in the growing point. Evidently, this elongation is accompanied by the unwinding of the parental double helix to expose a region of single-stranded DNA which is then converted to the duplex state by a discontinuous mechanism involving the synthesis of DNA fragments.     

Bacteriophage T7 gene 2.5 protein: an essential protein for DNA replication.

The product of gene 2.5 of bacteriophage T7, a single-stranded DNA binding protein, physically interacts with the phage-encoded gene 5 protein (DNA polymerase) and gene 4 proteins (helicase and primase) and stimulates their activities. Genetic analysis of T7 phage defective in gene 2.5 shows that the gene 2.5 protein is essential for T7 DNA replication and growth. T7 phages that contain null mutants of gene 2.5 were constructed by homologous recombination. These gene 2.5 null mutants contain either a deletion of gene 2.5 (T7 delta 2.5) or an insertion into gene 2.5 and cannot grow in Escherichia coli (efficiency of plating, < 10(-8)). After infection of E. coli with T7 delta 2.5, host DNA synthesis is shut off, and phage DNA synthesis is reduced to < 1% of phage DNA synthesis in wild-type T7-infected E. coli cells as measured by incorporation of [3H]thymidine. In contrast, RNA synthesis is essentially normal in T7 delta 2.5-infected cells. The defects in growth and DNA replication are overcome by wild-type gene 2.5 protein expressed from a plasmid harboring the T7 gene 2.5.     

Bacteriophage T3 and T7 early RNAs are translated by eukaryotic 80S ribosomes: active phage T3 coded S-adenosylmethionine cleaving enzyme is synthesized.

RNA transcribed in vitro from the early region of bacteriophage T3 or T7 was translated by cytoplasmic ribosomes which synthesized protein in cell-free systems prepared from mammalian cells and wheat germ. The proteins synthesized in vitro and their counterparts prepared from infected Escherichia coli comigrate by polyacrylamide gel electrophoresis with sodium dodecyl sulfate and are similarly affected by deletion or amber bacteriophage mutations. Bacteriophage T3 codes for an enzyme that cleaves S-adenosylmethionine and this activity was detected among the products of the mammalian cell-free system. Bacteriophage T3 or T7 RNA, after endoribonuclease III (EC 3.1.4.24) cleavage, gave higher levels of incorporation into phage T3 or T7 polypeptides than when an equivalent amount of the uncleaved RNA was added to the eukaryotic cell-free systems. Methylation of phage T3 or T7 RNAs is apparently not required for translation in either the wheat germ or mammalian cell-free system. The ability of T3 and T7 RNA to be translated in the presence of saturating amounts of natural eukaryotic mRNAs suggests that many prokaryotic genes introduced into mammalian cells might be expressed if they were transcribed in an appropriate form.     

In Vitro Synthesis of Deoxynucleotide Kinase Programmed by Bacteriophage T4-RNA

RNA extracted from T4 phage-infected Escherichia coli cells can direct the synthesis of phage-specific deoxynucleotide kinase in a cell-free system from uninfected E. coli. The maximum messenger activity is obtained 8 min after infection at 37 degrees C and decreases there-after. The main activity of this messenger RNA has a sedimentation coefficient of about 15 S on a sucrose density gradient. The conditions necessary for the appearance of enzyme activity in vitro are the same as those required for de novo synthesis of protein. RNA from cells infected with a T4 amber mutant incapable of inducing the kinase in vivo lacks the ability to direct the synthesis of enzyme in vitro.     

Resistance to Colicins E3 and K Induced by Infection with Bacteriophage f1

When infected with the filamentous phage f1, Escherichia coli K-12 strains are 20- to 40-times more resistant to colicins K and E3 than are uninfected cells. The colicins are adsorbed normally and are blocked in their action at some subsequent stage of their action. Productive infection of virions is not necessary for induction of resistance. The evidence presented implicates the product of gene III of the phage, a minor protein in the virion, as being responsible for this resistance. The simplest interpretation of the results is that this gene product blocks the penetration of colicins which is necessary for their action.     

Suppression of Chemical Mutagenesis in Bacteriophage T4 by Genetically Modified DNA Polymerases

Two antimutagenic DNA polymerases of bacteriophage T4 markedly reduce transition mutagenesis by a variety of chemical mutagens. Spontaneous mutation and mutagenesis by 2-aminopurine, 5-bromodeoxyuridine, and thymine deprivation are strongly suppressed. Mutagenesis at G:C sites by ethyl methanesulfonate, and at A:T sites by nitrous acid, is moderately suppressed. Mutagenesis at G:C sites by hydroxylamine and by nitrous acid is not suppressed. These results support the notion that the indispensable DNA polymerase of bacteriophage T4 plays a crucial role in the selection of the correct base during DNA replication. The data also reveal that mutagenic specificities of chemical agents depend as much upon the characteristics of the enzymatic apparatus of DNA replication as they do upon the chemistry of primary mutational lesions.     

Detection of Bacteriophage T4- and T5-Coded Transfer RNAs

A hybridization procedure using mixtures of radioactive aminoacyl-tRNA is described for detecting new phage-induced tRNA species. Five phage-coded tRNA species have been identified from T4 phage infected bacteria and 14 from T5 phage infected cells.     

Structure of the Replicating DNA from Bacteriophage T4

At an early stage of replication, parental T4 DNA shows a loop structure often displaying two 3'-ended, single-stranded "whiskers", located in trans configuration at the branching-points. Several such loops have been observed within a single T4 molecule. Occasionally, reinitiation occurred in the middle of a loop, which suggests that the loop was growing in both directions.     

Self-Association of Gene-32 Protein of Bacteriophage T4

The self-association of gene-32 protein has been studied by sedimentation equilibrium centrifugation and polyacrylamide gel electrophoresis, in order to better understand its role in DNA replication and genetic recombination. The monomer molecular weight of gene-32 protein is 38,000 in guanidine hydrochloride and 34,000 in sodium dodecyl sulfate, in agreement with the results of Alberts and coworkers. Stable dimers of gene-32 protein occur under various conditions, among which are high ionic strength and pH 10. The occurrence of stable dimers under some conditions and higher aggregates under others indicates there are two types of protein-protein interactions occurring in gene-32 protein self-association. The association that occurs above about 0.1 mg/ml concentration of protein produces at least decamers.A model for the DNA replication fork is postulated that requires the two different interactions that occur in gene-32 protein aggregation. In the model, gene-32 protein holds the two strands of the DNA duplex in a conformation that prevents their reannealing and, therefore, facilitates replication and recombination.     

Bacteriophage T4 polynucleotide kinase triggers degradation of mRNAs

The bacteriophage T4-encoded RegB endoribonuclease is produced during the early stage of phage development and targets mostly (but not exclusively) the Shine-Dalgarno sequences of early genes. In this work, we show that the degradation of RegB-cleaved mRNAs depends on a functional T4 polynucleotide kinase/phosphatase (PNK). The 5'-OH produced by RegB cleavage is phosphorylated by the kinase activity of PNK. This modification allows host RNases G and E, with activity that is strongly stimulated by 5'-monophosphate termini, to attack mRNAs from the 5'-end, causing their destabilization. The PNK-dependent pathway of degradation becomes effective 5 min postinfection, consistent with our finding that several minutes are required for PNK to accumulate after infection. Our work emphasizes the importance of the nature of the 5' terminus for mRNA stability and depicts a pathway of mRNA degradation with 5'- to 3'-polarity in cells devoid of 5'-3' exonucleases. It also ascribes a role for T4 PNK during normal phage development.