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Index >> Bacteriophages - Part One >> Formation of Replicative Form ( SS to RF )
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Formation of Replicative Form ( SS to RF )


Formation of Replicative Form ( SS to RF ) - Single stranded (SS) viral DNA is converted into the duplex replicative form (RF) on the inner membrane of the bacterial cell. During this process the (+) strand serves as a template for the synthesis of a complementary (-) strand. The formation of the RF form is essentially similar to that of φX 174, except for the initiation process.


It has been suggested that the gene III protein attaches the infecting viral strand to the inner cell membrane, or possibly to a cellular replicative system associated with the membrane ssDNA-RF' formation in M 13 is prevented by rifampicin (an inhibitor of E. coli RNA polymerase), but not by chloramphenicol (protein synthesis inhibitor).


This indicates that RNA polymerase is required for the formation of parental RF, but not for phage specified protein synthesis. The host RNA polymerase lays down a short complementary RNA primer on the viral strand within the intergenic space.


The formation of the, M13 primer is catalysed by a different form of RNA polymerase, called RNA polymerase Ill. This can be separated from the classic RNA polymerase I by gel filtration. It appears to contain a small subunit which enables it to differentiate between M 13 and φX 174 DNA templates.

RNA polymerase III is assayed by its ability, in the presence of unwinding protein to prime DNA polymerase III holoenzyme replication of Ml3 DNA but not of φXI74 DNA. This discrimination depends upon the coating of the template single strand with unwinding protein.


In the absence of the unwinding protein both M13 and φX174 DNA can be primed by RNA polymerase III. RNA polymerase III has only a feeble capacity for transcribing dsDNA  (less than 5% of the specific activity of DNA polymerase I).

Treatment of RNA polymerase III with rifampicin inactivates the enzyme, and causes the release of the small subunit. This factor, if added to RNA polymerase I, gives it the latter the capacity to distinguish between MI3 and φXI74 templates.

The DNA unwinding protein extends the viral DNA molecule. It may help in the correct selection of the initiation site, perhaps by masking other initiation sites.

The DNA chain (-) is extended on the RNA primer, with the viral (+) strand serving as the template. The reaction is catalysed by the DNA polymerase III holoenzyme. This consists of a 140,000 dalton DNA polymerase III* polypeptide and a 77.000 dalton DNA copolymerase III* polypeptide. Copolymerase III* is require( for the formation of the initiation complex, but not for replication.

It thus appears to be analogous with the subunit of the RN A polymerase holoenzyme. As a result of the synthesis of the DNA complementary (-) strand an RFII DNA molecule is formed.

This molecule contains a gap at a unique site in the complementary strand. Conversion of the gapped RFII to the covalently closed duplex circle (RFI) is brought about by the combined action of E. coli DNA polymerase I and ligase.
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