Plasmid copy number:

• The copy number plasmid means the number of plasmids that are normally present in a single bacterial cell.
• The size and copy number of a plasmid is important.
• Cloning is concerned with it.
• Plasmid size that less than 10 kb is desirable for a cloning vector.
• Some plasmids, especially the larger ones, are stringent.
• Larger plasmids and have a low copy number which might be just one or two per cell.
• Others are called relaxed plasmids which are present in multiple copies of 50 or more per cell.
• Large quantities of the recombinant DNA molecule can be obtained when multiple copies of the cloning vector are present in the cell.
• Naturally occurring plasmids are usually stably maintained in their bacterial hosts.
• This stability often must be accomplished despite a very low number of plasmid copies per cell.

Plasmid replication and control:

• Many plasmids are replicated as double-stranded circular molecules.
• The replication of the double-stranded molecule is the same as that of the chromosome.
• Replication starts from the origin of replication which is known as
• It may be unidirectional ( single directional ) or bidirectional ( both directions).
• When the whole circle is completed, then it segregates.
• However, there are some aspects of replication that differ from that of the chromosome, especially for the multicopy plasmids.

Mechanism of regulation of plasmid copy number

• Regulation of plasmid number is important to control the copy number of the plasmid.
• The mechanisms used by some plasmids to regulate their copy number are :

1) ColE1-derived Plasmids:

• It is the regulation of the processing of primer by complementary RNA
• The mechanism of copy number regulation of the plasmid ColE1 was one of the first to be studied.
• The mechanism of regulation of ColE1-derived plasmids is shown in:
  • Replication starts always from the origin of replication (oriV)
  • RNA II forms the primer for the plasmid DNA replication.
  • At the origin of replication, an RNA-DNA hybrid is formed.
  • RNA is RNA II and that DNA is the DNA of plasmid.
  • Then RNase cleaves the RNA II.
  • RNase H has endonuclease activity which means it can capacity to cleave.
  • At the cleaved or cut portion, 3 ʹOH is exposed where the nucleotides will be added by DNA polymerase I.
  • Thus the plasmid is replicated.
  • But when there is a sufficient number of plasmid its replication needs to be stopped.
  • This process is regulated by the RNA I and the Rop protein.
  • The Rop protein (sometimes called Rom)  helps RNA I to pair with RNA II.
  • RNA I is transcribed from the opposite strand to RNA II and is complementary to the first 108 bases of RNA II.
  • When there is pairing between RNA I and RNA II, RNAse can not cut the strand.
  • When the strand isn’t cleaved further replication is halted.
  • Therefore, it inhibits DNA replication.
  • The inhibition of replication is almost complete when the concentration of the plasmid reaches about 16 copies per cell which is the copy number of the original ColE1 plasmid.
  • Mutations that inactivate Rop cause only a moderate increase in the plasmid copy number.

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2. R1 plasmid:

• R1 plasmid is a member of the IncFII family of plasmids.
• Plasmids belonging to the INFII group (R1, R6-5, R100) are self-transmissible, conjugative plasmids found in Enterobacteriaceae.
• R1 plasmid regulates its copy number by regulating the amount of the Rep protein.
• Plasmid requires the Rep protein to initiate the replication.
• It is different from ColE1-derived plasmids which do not require Rep protein. Only the RNA primer was enough for it to start the replication process.
• The Rep protein is required to separate the strands of DNA at the oriV region, often with the help of host proteins, including DnaA.
• Opening the strands is a necessary first step of replication.
• The Rep proteins bind specifically in the DSO of DNA
• By controlling the synthesis of the Rep protein, the plasmid copy number can be controlled.
• The R1 plasmid uses its complementary RNA to inhibit the translation of the mRNA that encodes the Rep protein and inhibits the replication of the plasmid DNA.

• The plasmid-encoded protein RepA is the only plasmid-encoded protein that is required for the initiation of replication.
• Two promoters transcribe the repA gene:
  • pcopB
  • pCopA

CopB:

• CopB, codes for a protein that represses transcription of the repA gene.
• When the plasmid first enters a bacterial cell, the absence of CopB allows expression of RepA and so there is a burst of replication until the level of CopB builds up to repress this promoter.
• From then on, expression of RepA occurs at a low level from the copB promoter

CopA:

• The second regulatory gene, copA, then regulates the expression of RepA.
• This gene codes for an 80–90-nucleotide untranslated RNA molecule.
• The copA gene is within the region of DNA that is transcribed for the production of RepA but is transcribed in the opposite direction (it is an antisense RNA).
• The copA RNA is therefore complementary to a short region of the repA transcript and will bind to it, interfering with the translation of the RepA protein.
• When the plasmid replicates, the number of copies of the copA gene is doubled and the amount of the copA RNA will therefore increase.
• This causes a marked reduction in further replication initiation, until cell division

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