N15: the linear phage-plasmid

The lambdoid phage N15 of Escherichia coli is very unusual among temperate phages in that its prophage is not integrated into chromosome but is a linear plasmid molecule with covalently closed ends. Upon infection the phage DNA circularises via cohesive ends, then phage-encoded enzyme, protelomerase, cuts at an inverted repeat site and forms hairpin ends (telomeres) of the linear plasmid prophage. Replication of the N15 prophage is initiated at an internally located ori site and proceeds bidirectionally resulting in formation of duplicated telomeres. Then the N15 protelomerase cuts duplicated telomeres generating two linear plasmid molecules with hairpin telomeres. Stable inheritance of the plasmid prophage is ensured by partitioning operon similar to the F factor sop operon. Unlike F sop, the N15 centromere consists of four inverted repeats dispersed in the genome. The multiplicity and dispersion of centromeres are required for efficient partitioning of a linear plasmid. The centromeres are located in N15 genome regions involved in phage replication and control of lysogeny, and binding of partition proteins at these sites regulates these processes. Two N15-related lambdoid Siphoviridae phages, φKO2 in Klebsiella oxytoca and pY54 in Yersinia enterocolitica, also lysogenize their hosts as linear plasmids, as well as Myoviridae marine phages VP882 and VP58.5 in Vibrio parahaemolyticus and ΦHAP-1 in Halomonas aquamarina. The genomes of all these phages contain similar protelomerase genes, lysogeny modules and replication genes, as well as plasmid-partitioning genes, suggesting that these phages may belong to a group diverged from a common ancestor.     

The protelomerase of the phage-plasmid N15 is responsible for its maintenance in linear form

The prophage of coliphage N15 is not integrated into the bacterial chromosome but exists as a linear plasmid molecule with covalently closed ends. Upon infection of an Escherichia coli cell, the phage DNA circularises via cohesive ends. A phage-encoded enzyme, protelomerase, then cuts at another site, telRL, and forms hairpin ends (telomeres). We demonstrate that this enzyme acts in vivo on specific substrates, and show that it is necessary for replication of the linear prophage. We show that protelomerase is an end-resolving enzyme responsible for processing of replicative intermediates. Removal of protelomerase activity resulted in accumulation of replicative intermediates that were found to be circular head-to-head dimers. N15 protelomerase and its target site constitute a functional unit acting on other replicons independently of other phage genes; a mini-F or mini-P1 plasmid carrying this unit replicates as a linear plasmid with covalently closed ends. Our results suggest the following model of N15 prophage DNA replication. Replication is initiated at an internal ori site located close to the left end of plasmid DNA and proceeds bidirectionally. After replication of the left telomere, protelomerase cuts this sequence and forms two hairpin loops telL. After duplication of the right telomere (telR) the same enzyme resolves this sequence producing two linear plasmids. Alternatively, full replication of the linear prophage to form a circular head-to-head dimer may precede protelomerase-mediated formation of hairpin ends.     

The antirepressor needed for induction of linear plasmid-prophage N15 belongs to the SOS regulon

The physiological conditions and molecular interactions that control phage production have been studied in only a few families of temperate phages. We investigated the mechanisms that regulate activation of lytic development in lysogens of coliphage N15, a prophage that is not integrated into the host chromosome but exists as a linear plasmid with covalently closed ends. We identified the N15 antirepressor gene, antC, and showed that its product binds to and acts against the main phage repressor, CB. LexA binds to and represses the promoter of antC. Mitomycin C-stimulated N15 induction required RecA-dependent autocleavage of LexA and expression of AntC protein. Thus, a cellular repressor whose activity is regulated by DNA damage controls N15 prophage induction.     

Dual control of lysogeny by bacteriophage P22: An antirepressor locus and its controlling elements

Two distantly linked clusters of genes on the Salmonella typhimurium phage P22 chromosome are involved in the control of lysogeny and superinfection immunity. One cluster consists of genes c1, c2, and c3, which are primarily responsible for the establishment and maintenance of lysogeny. It has been proposed that the second cluster consists of three loci which are responsible for the synthesis and control of an antirepressor substance which overcomes the repression mediated by the c2 gene product. This paper reports the isolation of mutants in a locus designated “ant” having characteristics expected of antirepressor mutants. Evidence is presented that the other loci in this second immunity region, mnt and virA, control the expression of the ant gene as represser and promoter/operator, respectively. The interactions of these three loci with each other and with the other immunity region are discussed.     

Remote control of mRNA cleavage by a small RNA

Comment on: Prévost K, et al. Genes Dev 2011; 25:385-96.     

Translational control by delayed RNA folding: identification of the kinetic trap

The maturation or A-protein gene of single-stranded RNA phage MS2 is preceded by a 130-nt long untranslated leader. When MS2 RNA folding is at equilibrium, the gene is untranslatable because the leader adopts a well-defined cloverleaf structure in which the Shine-Dalgarno (SD) sequence of the maturation gene is taken up in long-distance base pairing with an upstream complementary sequence (UCS). Synthesis of the A-protein takes place transiently while the RNA is synthesized from the minus strand. This requires that formation of the inhibitory cloverleaf is slow. In vitro, the folding delay was on the order of minutes. Here, we present evidence that this postponed folding is caused by the formation of a metastable intermediate. This intermediate is a small local hairpin that contains the UCS in its loop, thereby preventing or slowing down its pairing with the SD sequence. Mutants in which the small hairpin could not be formed made no detectable amounts of A-protein and were barely viable. Apparently, here the cloverleaf formed quicker than ribosomes could bind. On the other hand, mutants in which the small intermediary hairpin was stabilized produced more A-protein than wild type and were viable. One hardly growing mutant that could not form the metastable hairpin and did not make detectable amounts of A-protein was evolved. The emerging pseudo-revertant had selected two second site repressor mutations that allowed reconstruction of a variant of the metastable intermediate. The pseudo-revertant had also regained the capacity to produce the A-protein.     

Characterization of the hCTR1 gene: genomic organization, functional expression, and identification of a highly homologous processed gene

The human hCTR1 gene was originally identified by its ability to complement a yeast mutant deficient in high-affinity copper uptake (Zhou, B., Gitschier, J., 1997. A human gene for copper uptake identified by complementation in yeast. Proc. Natl. Acad. Sci. USA 94, 7481-7486). Here, we have determined the DNA sequence of the exon-intron borders of the hCTR1 structural gene and report that the coding sequence is disrupted by three introns, all of which comply with the GT/AG rule. Furthermore, human fibroblasts, transfected with hCTR1 cDNA, were shown to have a dramatically increased capacity for (64)Cu uptake, indicating that the hCtr1 protein is functional in copper uptake in human cells. In contrast, no evidence was found for involvement of the hCTR2 gene product in copper uptake. Finally, we have identified a highly homologous processed pseudogene, hCTR1psi, which was localized to chromosome 3q25/26. The processed gene was found to be transcribed, but due to a frame shift mutation, it only had the potential to encode a truncated protein of 95 amino acid residues, and cells transfected with hCTR1psi DNA showed no increase of (64)Cu uptake.     

The gene for a small stable RNA (10Sa RNA) of Escherichia coli

A gene that codes for a small stable RNA (362 nucleotides) has been sequenced. It is a monocistronic gene, with its own promoter and terminator. It produces a precursor that is about 100 nucleotides longer than the mature RNA with all the extra nucleotides at the 3' end. The gene contains an open reading frame that corresponds to a small protein 25 amino acids long.     

Translational control by a small RNA: dendritic BC1 RNA targets the eukaryotic initiation factor 4A helicase mechanism

Translational repressors, increasing evidence suggests, participate in the regulation of protein synthesis at the synapse, thus providing a basis for the long-term plastic modulation of synaptic strength. Dendritic BC1 RNA is a non-protein-coding RNA that represses translation at the level of initiation. However, the molecular mechanism of BC1 repression has remained unknown. Here we identify the catalytic activity of eukaryotic initiation factor 4A (eIF4A), an ATP-dependent RNA helicase, as a target of BC1-mediated translational control. BC1 RNA specifically blocks the RNA duplex unwinding activity of eIF4A but, at the same time, stimulates its ATPase activity. BC200 RNA, the primate-specific BC1 counterpart, targets eIF4A activity in identical fashion, as a result decoupling ATP hydrolysis from RNA duplex unwinding. In vivo, BC1 RNA represses translation of a reporter mRNA with 5' secondary structure. The eIF4A mechanism places BC RNAs in a central position to modulate protein synthesis in neurons.     

Autocidal control of ticks by silencing of a single gene by RNA interference

Ticks impact human and animal health worldwide and new control methods are needed to circumvent drawbacks of tick control by acaricide application including selection of drug resistant ticks and environmental pollution. Using RNA interference we silenced the expression of a single gene, subolesin, and produced ticks with diminished reproductive performance and prevented successful mating and production of viable offspring. We propose a sterile acarine technique (SAT) for reduction of tick populations by release of subolesin-silenced ticks. Conservation of subolesin among tick species suggests that SAT may be useful for control of many medically and economically important tick species.