Bacteriophage P4 is a defective phage that needs a helper phage to grow lytically, since it lacks structural genes and genes encoding host cell lysis functions. P4 has the capacity to derepress the unrelated prophage P2, or its relatives after infection, thereby getting access to the late functions of the helper. The P4egene product (E protein) mediates this process via a derepression mechanism.
In this work, the E protein is identified as a 10.8 kDa polypeptide, is shown to be the only P4 encoded protein required to derepress prophage P2, leading to in situ P2 DNA replication. The E has no DNA-binding activity. However it is able to interact with the P2 C repressor as shown in the yeast two-hybrid assay system. Analysis in a two-plasmid derepression system has shown that induction of the E results in a switch of P2 transcription from the lysogenic to the lytic mode. This implies that the E protein functions as an anti-repressor.
The P2 C repressor acts as a dimer. The E protein is also shown to be a dimer in vivo. The E protein only marginally affects dimerisation of C, but the presence of C enhances multimeric forms of E protein. Furthermore, the binding of the C protein to its operator is inhibited by E in vitro. This indicates that the anti-repressor function of E is mediated by the formation of multimeric complexes of E and C that interferes with binding of C to its operator.
The P2 sos mutation makes the P2 prophage resistant to derepression by P4. The Sos protein, which is shown not to interact with E, has an amino acid substitution of T67I and it is located within the presumed E binding epitope from 60 to 70 aa of the C-terminal part of the C protein. This supports the hypothesis that this part of the C protein is involved in interacting with the E protein.
By in vitro mutagenesis,esuppressor mutations that overcome the resistance of Sos are isolated. A double mutant, E D65V+F68S, is able to interact with Sos in the yeast two-hybrid system but unable to derepress prophage P2 in the presence of Sos in the two-plasmid system. A single mutation, E D65V, has however lost the capacity to derepress the prophage P2 from the lysogenic to the lytic mode in the two-plasmid system. This indicates that amino acid 65 in E is important for its interaction with the C repressor.
Moreover, P4 E can derepress the P2-related phage WFthat is heteroimmune to P2. The DNA sequences of the whole early control region of phage WFhave been determined. The transcriptional switch region of WFphage has a similar arrangement as those of P2, and P2-related phages186 and HP1. WFphage exploits similar strategy to control the lytic versus lysogenic development. The interaction between P4 and WF, however, is only one way, i.e. P4 is able to derepress WFphage but not the other way around.
Stockholm: Stockholm University , 1999. , 55 p.