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 MOBILITY.

Plasmid Host Range Refers to plasmids ability to survive/replicate in a particular host.

Some plasmids are able to replicate in a limited number of bacterial species; they have a NARROW host range.

Examples: ColE1, pBR322, pUC18 plasmids which are limited 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 IIThis 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.

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