Plasmid copy number
This article may be too technical for most readers to understand.(September 2015) |
In
Regulation
Medium copy number plasmids, also called relaxed plasmids, require a system to ensure that replication is inhibited once the number of plasmids in the cell reaches a certain threshold. Relaxed plasmids are generally regulated through one of two mechanisms: antisense RNA or iteron binding groups. Low copy number plasmids, also called stringent plasmids, require tighter control of replication.
ColE1 derived plasmids: Antisense RNA
In ColE1 derived plasmids, replication is primarily regulated through a small plasmid-encoded RNA called RNA I. A single promoter initiates replication in ColE1: the RNA II promoter. The RNA II transcript forms a stable RNA-DNA hybrid with the DNA template strand near the origin of replication, where it is then processed by RNaseH to produce the 3' OH primer that DNA polymerase I uses to initiate leading strand DNA synthesis. RNA I serves as a major plasmid-encoded inhibitor of this process whose concentration is proportional to plasmid copy number. RNA I is exactly complementary to the 5' end of the RNA II (because it is transcribed from the opposite strand of the same region of DNA as RNA II). RNA I and RNA IIled a kissing complex. The kissing complex is stabilized by a protein called Rop (repressor of primer) and a double-stranded RNA-I/RNA-II RNA duplex is formed. This altered shape prevents RNA II from hybridizing to the DNA and being processed from RNaseH to produce the primer necessary for initiation of plasmid replication. More RNA I is produced as the plasmid concentration increases, which increasingly inhibits replication, resulting in regulation of copy number.[2][3]
R1 and ColIb-p9 Plasmids: Antisense RNA
Most plasmids require a plasmid-encoded protein, usually called Rep, to separate the strands of DNA at the origin of replication (oriV) to initiate DNA replication. Rep binds to specific DNA sequences in oriV which are unique to a plasmid type. The synthesis of Rep protein is controlled in order to limit plasmid replication and therefore regulate copy number. In R1 plasmids RepA can be transcribed from two different promoters. It is made from the first promoter until the plasmid reaches its copy number, upon which the protein CopB represses this primary promoter.[3] RepA expression is also regulated post-transcriptionally from the secondary promoter by an antisense RNA called CopA. CopA interacts with its RNA target in the RepA mRNA and forms a kissing complex and then a RNA-RNA duplex. The resultant double stranded RNA is cleaved by RNase III, preventing synthesis of RepA. The higher the concentration of the plasmid, the more CopA RNA is produced and the less RepA protein can be synthesized, increasing inhibition of plasmid replication.[4]
Col1b-P9: Antisense RNA
Replication of the low-copy-number ColIb-P9 depends upon Rep, which is produced by expression of the repZ gene. repZ expression requires formation of a pseudoknot in the mRNA. repZ is repressed by a small antisense Inc RNA, which binds to repZ mRNA, forms an Inc RNA-mRNA duplex, and prevents formation of the pseudoknot to inhibit repZ translation into Rep. In this event, replication can no longer occur.[5]
pSC101: Iteron plasmid
Iteron plasmids, including
Incompatibility
Plasmids can be incompatible if they share the same replication control mechanism. Under these circumstances, both plasmids contribute to the total copy number and are regulated together. They are not recognized as distinct plasmids. As such, it becomes much more likely that one of the plasmids may be out-copied by the other and lost during cell division (the cell is "cured" of the plasmid).[3] This is particularly likely with low copy number plasmids. Plasmids can also be incompatible due to shared partitioning systems.