Plasmids Lecture 2

1
Plasmids (Lecture 2)

• Replication Schemes
• Initiation of Replication
• Control of Replication
• Amplification of Plasmid
• Plasmid Cure
• Plasmid Partition
• Incompatibility

2
Replication Schemes
oriV
A) Rolling-circle replication
One strand of DNA is nicked to provide 3OH for
DNA synthesis. No RNA primer is required.
Nick
3
Replication Schemes
B) Theta replication Unidirectional Bi-dire
ctional
oriV
oriV
q
Common form of replication.
4
Initiation at Replication Origin

• Opening of DNA duplex (Theta replication)
• RNA polymerase transcribes RNAII (555bases)
• 3-end transcript forms D loop (RNA-DNA-DNA
  loop)

oriV
oriV
RNA
5

• Primer formation
• origin defined by site cleaved by RNaseH
• Ribonuclease H is an endoribonuclease that
  specifically degrades the RNA strand in an
  RNA-DNA hybrid.
• cleaved RNA primer forms 3 end for DNA synthesis
• DNA Pol I may synthesize DNA or Pol III, DnaB and
  DnaG. Form a replisome.

DNA
Rnase H
3
oriV
RNA
oriV
6
Control of Plasmid Replication

• I. Regulation of primer synthesis by
  antisense RNA (e. g. pColE1)
• II. Regulation of essential replication
  proteins (e. g. pR1)
• III. Regulation of the replication proteins by
  binding to iterons (e.g. pSC101)

7
A) Control through RNA II
pColE1
RNA II a primer for DNA replication. RNA I
complementary to RNA II form duplex with RNA II.
Rop facilitates the pairing between RNA I and
RNA II.
8
When the number of plasmids is low inside cell
RNA II is largely produced to initiate
replication. When the number of plasmids is
increased inside cell A) More RNA I will be
synthesized. RNA I binds to RNAII to inhibit
replication. B) Production of Rop protein
increases the binding affinity between RNA I
and RNA II.
9
B) Control through RepA protein production
pR1
RepA protein replication protein. CopB
protein repressor of the gene repA
transcription. CopA RNA complementary to the
repA RNA. PrepA promoter of the repA gene.
PcopB promoter of both copB and repA genes.
10
When the number of plasmids is low inside
cell Transcription from PrepA produce RepA.
Little amounts of CopB protein is
produced. When the number of plasmids is high
inside cell A) CopB is accumulated and PrepA
is repressed by CopB. RepA can only be
transcribed from PcopB. Both RepA and CopB are
produced. B) More copA RNA is produced to
inhibit production of RepA through binding to
repA mRNA.
11
C) Control through RepA protein and iteron
pSC101
RepA Replication protein. Iteron DNA
sequence of plasmids that facilitates RepA
binding. (repeated 18-22 bp sites)
12
When the number of plasmids is low inside
cell RepA protein is an activator of
replication. When the number of plasmids is
high inside cell RepA represses its own
expression by binding to its own promoter.
High concentration of RepA will link two
plasmids by binding to the iteros of
plasmids and preventing replication. Couplin
g model
13
Plasmid Amplification

• Plasmid amplification
• - Artificially control the number of plasmid in
  cell.
• chloramphenicol inhibits protein synthesis
  Repressor can't be made so can't control the copy
  number, and plasmids can replicate to a high copy
  number.

14
Plasmid Cure

• Plasmid curing cells lose plasmids.
• Bacteria lose plasmids during cell division.
• Acridine orange specifically inhibits plasmids
  replication but not chromosomal replication.

15
Prevent Plasmid Cure during Cell Division
Partition

• Plasmids have a specific site called par that is
  involved in partition.
• Two models
• A) Cell membrane has specific sites for plasmids
  to bind.
• B) Genomic DNA has specific sites for plasmids to
  bind.

16
(No Transcript)
17
Incompatibility

• Plasmids sharing the same replication mechanism
  are incompatible.
• Using antibiotics to force both plasmids in the
  same cells.
• Plasmids sharing the same par function are
  incompatible.

18
Shuttle Plasmids

• Shuttle plasmid has two different replication
  origins that can replicate in different organisms.

oriVa
oriVa
oriVb
oriVb
Organism A
Organism B