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Plasmid Replication

Plasmids must be able to replicate independently of


the host chromosome. They have their own origin of
replication (ori).

There are two general modes of replication:


1) THETA Replication -- which is found in most of the
gram negative bacterial plasmids such as ColE1,
RK2, and F
2) ROLLINGCIRCLE Replication -- which is found in
some gram positive bacterial plasmids such pLS1,
pLB4
Replication is central to control number of important
plasmid properties:

HOST RANGE, COPY NUMBER, INCOMPATIBILITY and

Plasmid Host Range

Refers to plasmids ability to survive/replicate in a particu


host.

Some plasmids are able to replicate in a limited numbe


bacterial species; they have a NARROW host range.

Examples: ColE1, pBR322, pUC18 plasmids which are lim


to E.coli and some closely related species.
Other plasmids are able to replicate in a wide range of
bacterial species; they have a BROAD host range.
Eg: RSF1010 and RK2

Copy number control of


ColE1 Plasmid

The number of copies of a plasmid can vary from 1 ( F


plasmid) to 100 (pUC18).
Copy number is an important property of the plasmid
and depends on the mechanism by which it regulates
its own replication.
Replication control of ColE1 Plasmid
ColE1 is a small circular plasmid which codes for a 57
kDal protein toxin (colicin E1) which can kill other
E.coli cells.

It carries (oriV) origin of replication and a region


(bom) at which it can be mobilized for transfer to
other bacteria.

Other Genes of ColE1 Plasmid and their


functions as follows:

Circular Map of ColE1 Plasmid

Replication of ColE1 proceeds unidirectionally from


oriV and depends on RNA II which is transcribed from a
promoter 555 bp away from oriV.
When this transcript reaches oriV, one of two possible
fates is possible:
The RNA-DNA hybrid is cleaved by RNaseH to
generate a free 3'-OH end which serves as a primer for
DNA polymerase I to initiate replication.
Then DNA polymerase III takes over after a short
distance.

If RNA-DNA hybrid is NOT cleaved by RNaseH and


transcription continues nonproductively.
These fates depends on the formation of a specific
secondary structure in the nascent RNA II transcript;
If the structure forms correctly then RNaseH
processing occurs correctly.
If anything interfere with the formation of correct
secondary structure will interfere with replication

A specific RNA called RNA I whose function is to


interfere with the secondary structure of RNA II

This RNA I is a 108 nt molecule which is transcribed in the


opposite direction to RNA II from a promoter located
between the RNA II promoter and the origin of replication

Since RNA I is complementary to RNA II, it form a


stable RNA-RNA hybrid with it. If it does, then RNA II
will not be able to function as a primer.

This is an example of antisense RNA


regulation.

Replication of ColE1 also depends on the Rop


protein

Rop is a small protein (63 aas) which increases the


rate of binding of RNA I to RNA II. If Rop is absent,
plasmid replication will be more frequent.