Plasmid copy number

Last updated May 24, 2019

Plasmids must regulate their copy number (average number of plasmid copies per cell) to ensure that they do not excessively burden the host or become lost during cell division. Plasmids may be either high copy number plasmids or low copy number plasmids; the regulation mechanisms between these two types are often significantly different. Biotechnology applications may involve engineering plasmids to allow a very high copy number. For example, pBR322 is a low copy number plasmid (~20 copies/cell) from which several very high copy number cloning vectors (~1000 copies/cell) have been derived.^[1]

A plasmid is a small 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; however, plasmids are sometimes present in archaea and eukaryotic organisms. In nature, plasmids often carry genes that benefit the survival of the organism, such as by providing antibiotic resistance. While the chromosomes are big and contain all the essential genetic information for living under normal conditions, plasmids usually are very small and contain only additional genes that may be useful in certain situations or conditions. Artificial plasmids are widely used as vectors in molecular cloning, serving to drive the replication of recombinant DNA sequences within host organisms. In the laboratory, plasmids may be introduced into a cell via transformation.

Biotechnology Use of living systems and organisms to develop or make useful products

Biotechnology is the broad area of biology involving living systems and organisms to develop or make products, or "any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use". Depending on the tools and applications, it often overlaps with the (related) fields of molecular biology, bio-engineering, biomedical engineering, biomanufacturing, molecular engineering, etc.

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 postdoctoral researcher who constructed it. The p stands for "plasmid," and BR for "Bolivar" and "Rodriguez."

Regulation

High 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.

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 thereby blocks its translation into protein. asRNAs have been found in both prokaryotes and eukaryotes, antisense transcripts can be classified into short and long non-coding RNAs (ncRNAs). The primary function of asRNA is regulating gene expression. asRNAs may also be produced synthetically and have found wide spread use as research tools for gene knockdown. They may also have therapeutic applications.

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 include antisense RNAs and ctRNAs. Iterons complex with cognate replication (Rep) initiator proteins to achieve the required regulatory effect.

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 II first form a weak interaction called 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 duxplex 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 when the concentration of the plasmid is high, and high concentration of RNA I inhibits replication, resulting in regulation of copy number.^[2]^[3]

ColE1 is a plasmid found in bacteria. Its name derives from the fact that it carries a gene for colicin E1. It also codes for immunity from this product with the imm gene. In addition, the plasmid has a series of mobility (mob) genes. Its size and the presence of a single EcoRI recognition site caused it to be considered as a vector candidate.

RNAI is a non-coding RNA that is an antisense repressor of the replication of some E. coli plasmids, including ColE1. Plasmid replication is usually initiated by RNAII, which acts as a primer by binding to its template DNA. The complementary RNAI binds RNAII prohibiting it from its initiation role. The rate of degradation of RNAI is therefore a major factor in the control of plasmid replication. This rate of degradation is aided by the pcnB gene product, which polyadenylates the 3' end of RNAI targeting it for degradation by PNPase.

Transcription (biology) Process of copying a segment of DNA into RNA

Transcription is the first step of DNA based gene expression, in which a particular segment of DNA is copied into RNA by the enzyme RNA polymerase. Both DNA and RNA are nucleic acids, which use base pairs of nucleotides as a complementary language. During transcription, a DNA sequence is read by an RNA polymerase, which produces a complementary, antiparallel RNA strand called a primary transcript.

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]

The origin of replication is a particular sequence in a genome at which replication is initiated. This can either involve the replication of DNA in living organisms such as prokaryotes and eukaryotes, or that of DNA or RNA in viruses, such as double-stranded RNA viruses.

The R1 Plasmid is a plasmid that was first isolated from Salmonella paratyphi bacteria in 1963.

Post-transcriptional regulation is the control of gene expression at the RNA level, therefore between the transcription and the translation of the gene. It contributes substantially to gene expression regulation across human tissues.

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]

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 mosaic virus in 1982. Pseudoknots fold into knot-shaped three-dimensional conformations but are not true topological knots.

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.^[3] pSC101 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.^[3] 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.^[6]

The fertility factor allows genes to be transferred from one bacterium carrying the factor to another bacterium lacking the factor by conjugation. The F factor is carried on the F episome, the first episome to be discovered. Unlike other plasmids, F factor is constitutive for transfer proteins due to a mutation in the gene finO. The F plasmid belongs to a class of conjugative plasmids that control sexual functions of bacteria with a fertility inhibition (Fin) system.

The RK2 Plasmid is a broad-host-range plasmid belonging to the incP incompatibility group It is notable for its ability to replicate in a wide variety of single-celled organisms, which makes it suitable as a genetic engineering tool. It is capable of transfer, replication, and maintenance in most genera of Gram-negative bacteria. RK2 may sometimes be referred to as pRK2, which is also the name of another, unrelated plasmid. The IncP-1 plasmid group of which RK2 is a part has been described as "highly potent, self-transmissible, selfish DNA molecules with a complicated regulatory circuit"

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. They demonstrated that a gene from a frog could be transferred into bacterial cells and then expressed by the bacterial cells. The plasmid is a natural plasmid from Salmonella panama.

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.

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References

↑ Boros, I; Pósfai, G; Venetianer, P (October 1984). "High-copy-number derivatives of the plasmid cloning vector pBR322". Gene. 30 (1–3): 257–60. doi:10.1016/0378-1119(84)90130-6. PMID 6096220.
↑ Cesareni, G; Helmer-Citterich, M; Castagnoli, L (1991). "Control of ColE1 plasmid replication by antisense RNA". Trends in Genetics. 7 (7): 230–235. doi:10.1016/0168-9525(91)90370-6. PMID 1887504.
1 2 3 4 5 Snyder, Larry; Peters, Joseph E.; Henkin, Tina M.; Champness, Wendy (2013). Molecular Genetics of Bacteria (4th ed.). ASM Press. ISBN 978-1555816278.
↑ Blomberg, P; Nordström, K; Wagner, E. G. (1992). "Replication control of plasmid R1: RepA synthesis is regulated by CopA RNA through inhibition of leader peptide translation". The EMBO Journal. 11 (7): 2675–2683. PMC 556743 . PMID 1378398.
↑ Asano, K; Mizobuchi, K (1998). "Copy number control of IncIalpha plasmid ColIb-P9 by competition between pseudoknot formation and antisense RNA binding at a specific RNA site". The EMBO Journal. 17 (17): 5201–5213. doi:10.1093/emboj/17.17.5201. PMC 1170848 . PMID 9724656.
↑ Kunnimalaiyaan, S; Inman, R. B.; Rakowski, S. A.; Filutowicz, M (2005). "Role of π Dimers in Coupling ("Handcuffing") of Plasmid R6K's γ ori Iterons". Journal of Bacteriology. 187 (11): 3779–3785. doi:10.1128/JB.187.11.3779-3785.2005. PMC 1112066 . PMID 15901701.

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[1] Boros, I; Pósfai, G; Venetianer, P (October 1984). "High-copy-number derivatives of the plasmid cloning vector pBR322". Gene. 30 (1–3): 257–60. doi:10.1016/0378-1119(84)90130-6. PMID 6096220.

[2] Cesareni, G; Helmer-Citterich, M; Castagnoli, L (1991). "Control of ColE1 plasmid replication by antisense RNA". Trends in Genetics. 7 (7): 230–235. doi:10.1016/0168-9525(91)90370-6. PMID 1887504.

[snyder-3] 1 2 3 4 5 Snyder, Larry; Peters, Joseph E.; Henkin, Tina M.; Champness, Wendy (2013). Molecular Genetics of Bacteria (4th ed.). ASM Press. ISBN 978-1555816278.

[4] Blomberg, P; Nordström, K; Wagner, E. G. (1992). "Replication control of plasmid R1: RepA synthesis is regulated by CopA RNA through inhibition of leader peptide translation". The EMBO Journal. 11 (7): 2675–2683. PMC 556743 . PMID 1378398.

[5] Asano, K; Mizobuchi, K (1998). "Copy number control of IncIalpha plasmid ColIb-P9 by competition between pseudoknot formation and antisense RNA binding at a specific RNA site". The EMBO Journal. 17 (17): 5201–5213. doi:10.1093/emboj/17.17.5201. PMC 1170848 . PMID 9724656.

[6] Kunnimalaiyaan, S; Inman, R. B.; Rakowski, S. A.; Filutowicz, M (2005). "Role of π Dimers in Coupling ("Handcuffing") of Plasmid R6K's γ ori Iterons". Journal of Bacteriology. 187 (11): 3779–3785. doi:10.1128/JB.187.11.3779-3785.2005. PMC 1112066 . PMID 15901701.