The ban operon of bacteriophage P1. Localization of the promoter controlled by P1 repressor

Repression of a strong promoter localized 5' to the P1 ban gene is a prerequisite for cloning the ban operon in the multicopy plasmid pBR325. Repression is brought about by the binding of P1 repressor to the operator of the ban operon (Heisig, A., Severin, I., Seefluth, A. K., and Schuster, H. (1987) Mol. Gen. Genet. 206, 368-376). Binding of RNA polymerase in vitro overlaps with the operator and is inhibited by P1 repressor as shown by electron microscopy. The mutant P1 bac, which renders ban expression constitutive, contains a single base pair exchange within the operator. As a consequence, more repressor is required (i) for the inhibition of binding of RNA polymerase, and (ii) for the electrophoretic retardation of a P1 bac DNA fragment when compared to the corresponding bac+ fragment. A P1 ban recombinant plasmid containing a 4-base pair deletion close to the operator still allows binding of repressor but not of RNA polymerase. By that means, a repressible promoter is located at the P1 map position 72 in a distance of about 2.5 kilobase pairs to the beginning of the ban gene.     

The c1 repressor of bacteriophage P1 operator-repressor interaction of wild-type and mutant repressor proteins.

The c1 repressor gene of bacteriophage P1 and the temperature-sensitive mutants P1c1.100 and P1c1.162 was cloned into an expression vector and the repressor proteins were overproduced. A rapid purification procedure was required for the isolation of the thermolabile repressor proteins. Identification of the highly purified protein of an apparent molecular weight of 33,000 as the product of the c1 gene was verified by (i) the coincidence of partial amino acid sequences determined experimentally to that deduced from the c1 DNA sequence, and (ii) the temperature-sensitive binding to the operator DNA of the thermolabile repressor proteins. Analysis of the products of c1-c1.100 recombinant DNAs relates the thermolability to an unknown alteration in the C-terminal half of the c1.100 repressor. Binding to the operator DNA of c1 repressor is sensitive to N-ethylmaleimide. Since the only three cysteine residues are located in the C-terminal half of the repressor it is suggested that this part of the molecule is important for the binding to the operator DNA. This assumption is supported by the findings that a 14-kDa C-terminal repressor fragment obtained by cyanogen bromide cleavage retains DNA binding properties.     

Immunity repressor of bacteriophage P2: Identification and DNA-binding activity

The product of gene C of the temperate bacteriophage P2, the immunity repressor, can be detected as a unique band eluting from phosphocellulose columns at 0.12 m-potassium phosphate when differentially labelled with a radioactive amino acid: the band is absent when phages that either have lost gene C through deletion or carry a suppressor-sensitive mutation in the gene are used. The repressor in its monomeric form is about 11,000 in molecular weight. At near physiological salt concentrations, the form predominantly recovered is the dimer.In filter-binding assays, the partially purified repressor binds wild-type P2 DNA strongly. It does not bind DNA of P2 vir94, a deletion that removes all the genetic elements involved in the regulation of lysogeny; it also does not bind, or binds inefficiently, DNA of P2 vir3, a mutation in the operator that controls the early replicative functions of P2. At the concentrations employed, the dimer is the active form in binding.The P2 repressor clearly differs in several features from the well-studied immunity repressor of bacteriophage lambda.     

Hexamer of bacteriophage f2 coat protein as a repressor of bacteriophage RNA polymerase synthesis.

Formation of complex I between phage f2 RNA and coat protein, leading to repression of phage RNA polymerase synthesis, depends nonlinearly upon the concentration of the coat protein. Maximum formation of complex I was observed when six molecules of coat protein were bound to one molecule of RNA. RNase digestion of a glutaraldehyde-fixed complex left, as the products, coat protein oligomers. The heaviest, hexamers, predominated in the mixture. It was also shown that, in an ionic environment required for phage protein synthesis, coat protein at a concentration optimum for complex I formation exists in solution as a dimer. The results indicate that the translational repression of the RNA polymerase cistron is due to a cooperative attachment to phage template of three dimers of coat protein, forming a hexameric cluster on an RNA strand.     

The Bof protein of bacteriophage P1 exerts its modulating function by formation of a ternary complex with operator DNA and C1 repressor.

Bacteriophage P1 encodes several regulatory elements for the lytic or lysogenic response, which are located in the immC, immI, and immT regions. Their products are the C1 repressor of lytic functions with the C1 inactivator protein Coi, the C4 repressor of antirepressor synthesis and the modulator protein Bof, respectively. We have studied in vitro the interaction of the components of the immC and immT regions with C1-controlled operators using highly purified Bof, C1, and Coi proteins. Bof protein (M(r) = 9,800) does not interact with C1 repressor alone, but as shown by DNA mobility shift experiments, in the presence of C1 repressor Bof binds to all operators tested by forming a C1.Bof-operator DNA ternary complex. The effect of this complex formation was studied in more detail with the operator of the c1 gene. Here, Bof only marginally alters the C1 repressor footprint at Op99a,b, but nevertheless considerably influences the repressibility of the operator.promoter element: (i) the autoregulated c1 mRNA synthesis is further down-regulated and (ii) the ability of Coi protein to dissociate the C1.operator DNA complex is strongly inhibited. We suggest that Bof protein functions by modulating C1 repression of many widely dispersed operators on the prophage genome.     

The c1 repressor inactivator protein coi of bacteriophage P1. Cloning and expression of coi and its interference with c1 repressor function

The immC region of bacteriophage P1 contains the c1 repressor gene and its upstream region with four c1-controlled operators and four open reading frames. A c1 inactivator gene, coi, was defined by mutations in immC that suppress the virulence of the P1virC mutation. The exact location of the coi gene was not known (Scott, J.R. (1980) Curr. Top. Microbiol. Immunol. 90, 49-65). When a variety of P1 immC fragments were inserted into an expression vector, a gene product was inducible for the open reading frame 4 only. We identify this product as the c1 inactivator protein, coi by the following criteria: (a) expression of coi from a recombinant plasmid induces the P1 prophage and inhibits lysogenization of sensitive bacteria by P1; (b) all c1-controlled operator-promoter elements tested in vivo are derepressed by coi; (c) a partially purified coi protein (apparent molecular weight = 4800) interacts with c1 repressor and inhibits its binding to the operator in vitro. Based on these results we refine a model for the regulation of those genes and elements within immC which participate in the decision of P1 to enter the lytic or lysogenic pathway.     

Programmed translational frameshift in the bacteriophage P2 FETUD tail gene operon

The major structural components of the P2 contractile tail are encoded in the FETUD tail gene operon. The sequences of genes F(I) and F(II), encoding the major tail sheath and tail tube proteins, have been reported previously (L. M. Temple, S. L. Forsburg, R. Calendar, and G. E. Christie, Virology 181:353-358, 1991). Sequence analysis of the remainder of this operon and the locations of amber mutations Eam30, Tam5, Tam64, Tam215, Uam25, Uam77, Uam92, and Dam6 and missense mutation Ets55 identified the coding regions for genes E, T, U, and D, completing the sequence determination of the P2 genome. Inspection of the DNA sequence revealed a new open reading frame overlapping the end of the essential tail gene E. Lack of an apparent translation initiation site and identification of a putative sequence for a programmed translational frameshift within the E gene suggested that this new reading frame (E') might be translated as an extension of gene E, following a -1 translational frameshift. Complementation analysis demonstrated that E' was essential for P2 lytic growth. Analysis of fusion polypeptides verified that this reading frame was translated as a -1 frameshift extension of gpE, with a frequency of approximately 10%. The arrangement of these two genes within the tail gene cluster of phage P2 and their coupling via a translational frameshift appears to be conserved among P2-related phages. This arrangement shows a striking parallel to the organization in the tail gene cluster of phage lambda, despite a lack of amino acid sequence similarity between the tail gene products of these phage families.     

The Mnt repressor of bacteriophage P22: role of C-terminal residues in operator binding and tetramer formation

A set of C-terminal deletion mutants of the Mnt repressor of bacteriophage P22 has been constructed, and the corresponding truncated proteins have been purified. A truncated protein lacking the three C-terminal residues, Lys80-Thr81-Thr82, binds operator DNA with an affinity near wild type and has a normal tetrameric structure. Loss of the next residue, Lys79, causes a 600-fold decrease in operator affinity, but the truncated protein is still tetrameric. Further sequential deletions of Tyr78 and Leu77 cause modest decreases in operator affinity, but the truncated proteins are now dimeric. These results indicate that Lys79 is an important determinant of the high affinity of Mnt repressor for operator DNA and that Tyr78 is an important determinant of tetramer formation by Mnt repressor.     

Cloning of the immunity repressor determinant of bacteriophage P2 in the pBR322 plasmid

Summary Through in vitro recombination of DNA restriction fragments, we have constructed a plasmid, which expresses in vivo the immunity repressor gene (C) of bacteriophage P2. A bacterial strain carrying such a plasmid showed a high level of P2 specific immunity. It was lysogenized normally by an infecting P2. but the frequency of spontaneous phage production was reduced about 10⁴ fold as compared to a normal P2 lysogen. Satellite phage P4, known to derepress P2 lysogens, was unable to derepress the plasmid-carrying lysogenic strain so to allow growth of coinfecting P2. Phage P4 multiplied on the plasmid-carrying. P2-lysogenic strain, but due to a prolonged latent period failed to form plaques on this strain.     

The old exonuclease of bacteriophage P2.

The Old protein of bacteriophage P2 is responsible for interference with the growth of phage lambda and for killing of recBC mutant Escherichia coli. We have purified Old fused to the maltose-binding protein to 95% purity and characterized its enzymatic properties. The Old protein fused to maltose-binding protein has exonuclease activity on double-stranded DNA as well as nuclease activity on single-stranded DNA and RNA. The direction of digestion of double-stranded DNA is from 5' to 3', and digestion initiates at either the 5'-phosphoryl or 5'-hydroxyl terminus. The nuclease is active on nicked circular DNA, degrades DNA in a processive manner, and releases 5'-phosphoryl mononucleotides.