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Drybench Plasmid.

The origin of replication and plasmid copy number

The origin is where a plasmid's replication starts, and in ColE1-type plasmids such as pBR322 and pUC19 it also decides how many copies each cell holds.

An RNA primer and an RNA brake

Replication of a ColE1-type plasmid starts from a transcript, RNA II, made upstream of the origin; once it is processed it primes DNA synthesis. A second, shorter transcript, RNA I, is made from the opposite strand of the same region and can pair with RNA II and stop it priming (Tomizawa and Itoh 1981).

Because RNA I acts on any plasmid with the same origin, two plasmids sharing an origin compete in one cell: they are incompatible, and one is usually lost without selection for both.

pBR322 and pUC19

pBR322 carries rop, whose product lowers the copy number (Cesareni et al. 1982). pUC plasmids lack rop and carry a point mutation in RNA II that raises the copy number further (Lin-Chao et al. 1992), which is one reason pUC plasmids give more DNA from a miniprep.

The map labels the region by RNA I, the part the annotation library takes from a submitter-annotated record of ColE1.

See it on a real plasmid

Open pBR322 and find rop and RNA I; then open pUC19, which has RNA I but no rop.

Open pBR322 in the plasmid mapSee it in the cell tour

RNA I and RNA II are transcripts: copy number is controlled by RNA, made as the tour's transcription step shows.

References

  1. Tomizawa J, Itoh T. Plasmid ColE1 incompatibility determined by interaction of RNA I with primer transcript. Proc Natl Acad Sci U S A 1981;78:6096-6100. PubMed 6171811
  2. Cesareni G, Muesing MA, Polisky B. Control of ColE1 DNA replication: the rop gene product negatively affects transcription from the replication primer promoter. Proc Natl Acad Sci U S A 1982;79:6313-6317. PubMed 6183660
  3. Lin-Chao S, Chen WT, Wong TT. High copy number of the pUC plasmid results from a Rom/Rop-suppressible point mutation in RNA II. Mol Microbiol 1992;6:3385-3393. PubMed 1283002
  4. Sutcliffe JG. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harb Symp Quant Biol 1979;43:77-90. PubMed 383387

Statements summarised by the site's builder from the references, for scientific review.