In
cellular biology
Cell biology (also cellular biology or cytology) is a branch of biology that studies the structure, function, and behavior of cells. All living organisms are made of cells. A cell is the basic unit of life that is responsible for the living and ...
, the plasmid copy number is the number of copies of a given
plasmid
A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; how ...
in a cell. To ensure survival and thus the continued propagation of the plasmid, they must regulate their copy number. If a plasmid has too high of a copy number, they may excessively burden their host by occupying too much cellular machinery and using too much energy. On the other hand, too low of a copy number may result in the plasmid not being present in all of their host's progeny. Plasmids may be either low, medium or high copy number plasmids; the regulation mechanisms between low and medium copy number plasmids are different. Low copy plasmids (5 or less copies per host) require either a
partitioning system or a toxin-antitoxin pair such as
CcdA/CcdB to ensure that each daughter receives the plasmid. For example, the F plasmid, which is the origin of BACs (
bacterial artificial chromosomes A bacterial artificial chromosome (BAC) is a DNA construct, based on a functional fertility plasmid (or F-plasmid), used for transforming and cloning in bacteria, usually '' E. coli''. F-plasmids play a crucial role because they contain partition ...
) is a single copy plasmid with a partitioning system encoded in an operon right next to the plasmid origin. The partitioning system interacts with the septation apparatus to ensure that each daughter receives a copy of the plasmid. Many
biotechnology
Biotechnology is the integration of natural sciences and engineering sciences in order to achieve the application of organisms, cells, parts thereof and molecular analogues for products and services. The term ''biotechnology'' was first used b ...
applications utilize mutated plasmids that replicate to high copy number. For example,
pBR322
pBR322 is a plasmid and was one of the first widely used '' E. coli'' cloning vectors. Created in 1977 in the laboratory of Herbert Boyer at the University of California, San Francisco, it was named after Francisco Bolivar Zapata, the postdoctora ...
is a medium copy number plasmid (~20 copies/cell) from which several high copy number
cloning vectors
A cloning vector is a small piece of DNA that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. The cloning vector may be DNA taken from a virus, the cell of a higher organism, or ...
(>100 copies/cell) have been derived by mutagenesis, such as the well known pUC series. This delivers the convenience of high plasmid DNA yields but the additional burden of the high copy number restricts the plasmid size. Larger high copy plasmids (>30kb) are disfavoured and also prone to size reduction through deletional mutagenesis.
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
Antisense RNA (asRNA), also referred to as antisense transcript, natural antisense transcript (NAT) or antisense oligonucleotide, is a single stranded RNA that is complementary to a protein coding messenger RNA (mRNA) with which it hybridizes, and ...
or
iteron
Iterons are directly repeated DNA sequences which play an important role in regulation of plasmid copy number in bacterial cells. It is one among the three negative regulatory elements found in plasmids which control its copy number. The others in ...
binding groups. Low copy number plasmids, also called stringent plasmids, require tighter control of replication.
ColE1 derived plasmids: Antisense RNA
In
ColE1
ColE1 is a plasmid found in bacteria. Its name derives from the fact that it carries a gene for colicin E1 (the ''cea'' gene). It also codes for immunity from this product with the ''imm'' gene. In addition, the plasmid has a series of mobility ...
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
DNA polymerase I (or Pol I) is an enzyme that participates in the process of prokaryotic DNA replication. Discovered by Arthur Kornberg in 1956, it was the first known DNA polymerase (and the first known of any kind of polymerase). It was initiall ...
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.
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
The origin of replication (also called the replication origin) is a particular sequence in a genome at which replication is initiated. Propagation of the genetic material between generations requires timely and accurate duplication of DNA by semi ...
(') to initiate DNA replication. Rep binds to specific DNA sequences in ' 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 plasmid The R1 Plasmid is a plasmid that was first isolated from '' Salmonella paratyphi'' bacteria in 1963. It is a short plasmid, composed of 97,566 nucleotides and 120 genes, that belongs to the IncFII plasmid group.
The R1 plasmid imparts multi-drug ...
s 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 protei
CopBrepresses this primary promoter.
RepA expression is also regulated
post-transcriptionally from the secondary promoter by an antisense RNA called
CopA
Copa or COPA may refer to:
COPA
COPA may refer to:
* Computer Operator Programming Assistant. trade of ITI
* Child Online Protection Act, a former U.S. law to protect minors from certain material on the internet
* Canadian Owners and Pilots Ass ...
. 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
Ribonuclease III (RNase III or RNase C)(BREND3.1.26.3 is a type of ribonuclease that recognizes dsRNA and cleaves it at specific targeted locations to transform them into mature RNAs. These enzymes are a group of endoribonucleases that are chara ...
, 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.
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
__NOTOC__
A pseudoknot is a nucleic acid secondary structure containing at least two stem-loop structures in which half of one stem is intercalated between the two halves of another stem. The pseudoknot was first recognized in the turnip yellow ...
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.
pSC101: Iteron plasmid
Iteron plasmids, including
F and
RK2-related plasmids, have
oriV regions containing multiple (~3-7) repeats of 17-22 bp iteron sequences.
pSC101
pSC101 is a DNA plasmid that is used as a cloning vector in genetic cloning experiments. pSC101 was the first cloning vector, used in 1973 by Herbert Boyer and Stanley Norman Cohen. Using this plasmid they have demonstrated that a gene from a fro ...
represents a simple model of an iteron plasmid. Iteron plasmids control copy number through two combined methods, suitable for low copy number stringent plasmids. One method is control of RepA synthesis. RepA is the only plasmid-encoded protein required for replication in pSC101. RepA protein represses its own synthesis by binding to its own promoter region and blocking transcription of itself (
transcriptional autoregulation). Thus, the more RepA is made, the more its synthesis is repressed, and subsequently limiting plasmid replication.
The coupling hypothesis proposes that the second method is coupling of plasmids through the Rep protein and iteron sequences. When the plasmid concentration is high, RepA plasmids bound to iterons form dimers in between two plasmids, "handcuffing" them at the origin of replication and inhibiting replication.
[{{cite journal, title=Role of π Dimers in Coupling ("Handcuffing") of Plasmid R6K's γ ori Iterons, journal=Journal of Bacteriology, date=2005, volume=187, issue=11, pages=3779–3785, pmc=1112066, pmid=15901701, doi=10.1128/JB.187.11.3779-3785.2005, last1=Kunnimalaiyaan, first1=S, last2=Inman, first2=R. B., last3=Rakowski, first3=S. A., last4=Filutowicz, first4=M]
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).
This is particularly likely with low copy number plasmids. Plasmids can also be incompatible due to shared
partitioning systems.
References
Plasmids