Linear plasmid ends

Plasmids
Extrachromosomal DNA that replicates separately from chromosome All have origin of replication (oriV) Encode proteins and RNA molecules Usually do not encode essential functions Usually have genes that confer an advantage under specific conditions Range in size from about 2 kb to several hundred kb Usually circular, but some are linear Negatively supercoiled like the chromosome Copy # per cell ranges from 1 - 100s and is controlled by the plasmid Have specific host range (not all plasmids replicate in all bacteria) Some can be transferred from one bacterium to another Certain plasmids are compatible with others (can exist stably in same cell) Can be easily purified from the cell Many plasmids have been altered to make them useful for moving foreign DNA in and out of a host cell (incorporating foreign DNA into a plasmid ensures that it will be replicated in the host) Plasmid naming: lowercase p; uppercase letters; numbers. e.g. pBR322

Circular plasmids

Most plasmids encode non-essential functions

Plasmid replication
Most plasmids use one of two common mechanisms of replication, both of which depend on the host cell replication machinery for replication (DNA polymerases, ligases, primases, helicases, etc. are usually supplied by the host cell): Theta replication - resembles replication of the chromosome and is named theta because the DNA resembles the greek letter ! during replication Strands of DNA are opened at the ori region An RNA primer is used to start replication Replication is either unidirectional (A) or bidirectional (B) Replication stops when the fork returns to the origin (uni- directional) or when two replication forks meet on the other side of the plasmid.

Plasmid replication
Rolling-circle replication involves a singlestranded intermediate that seems to roll off the plasmid. Rep protein nicks the plasmid at the double stranded ori DNA pol III uses the free 3-OH to replicate around the plasmid, displacing the non-template strand as it does so to give both double and single stranded versions of the plasmid The ends of the displaced strand are ligated to make a single stranded circular DNA molecule RNA polymerase makes a primer at the single-stranded origin (SSO) DNA pol III uses the RNA to prime replication around the single-stranded molecule DNA pol I replaces the RNA with DNA Ligase joins the two strands of DNA to make a second double- stranded plasmid

Regulation of copy number

Plasmid copy number can vary between 1 - 100s of copies per cell Relaxed plasmids have high copy number and regulate copy number loosely by inhibiting excessive replication Stringent plasmids have low copy number and regulate copy number more tightly ColE1 plasmids: Many of the most popular lab plasmids were derived from ColE1 or pMB1, which is very similar; eg. pBR322, pUC series, pBAD series, pACYC184 and the pET series. ColE1 plasmids use an antisense RNA called RNA I that interferes with processing of RNA II to the form that serves a primer to initiate replication by Pol I. The more copies of the plasmid, the higher the concentration of RNA I to inhibit replication. RNA I and RNA II are transcribed from the same region of DNA but in opposite directions using different, complementary template strands. Binding of RNA I to RNA II prevents it from binding to OriV being processed to its primer form by RNase H. The protein Rop stabilizes the interaction between RNAs I and II. Note that no plasmid-encoded protein is necessary for replication.

Regulation of copy number

Unilike ColEI, most plasmids require a plasmidencoded protein, called Rep, that separates the two strands of DNA at OriV to initiate replication. R1 plamids: R1 plasmids use an antisense RNA to inhibit synthesis of Rep protein, which is necessary for replication. The antisense RNA binds to and inhibits translation of an upstream orf to which the Rep gene is translationally coupled. The concentration of antisense RNA in the cell reflects the plasimd copy number. If the number of plasmids falls below the copy number, the lower antisense RNA concentration allows Rep to be translated and replication results.

Regulation of copy number

Iteron plasmids such as RK2, F and R6K use a protein

called RepA, which is necessary for replication, to regulate copy number in two ways. First, RepA negatively autoregulates its own transcription. The more copies of a plasmid in the cell, the more RepA transcription in repressed. Second, when a plasmid is in multicopy, RepA binds two of them together, which prevents replication instead of initiating it, as is the case when only one plasmid is bound. This method is used by some stringent plasmids.