General Properties of plasmids
- Replicons with characteristic copy level maintenance functions
- Extrachromosomal some interact with chromosome.
- Small 1.5 – 200kb + replicate v quickly (max 2-3 min)
- Non-essential for viability of host cell can confer selective advantage on host (Abr)
- May be transmissible between cells - by conjugation or mobilisation
what are the two Replication modes of plasmids?
- democratic All replicated once only
2. random choice mixture are replicated.
whats Plasmid Incompatibility?
inability of two plasmid species to coexist
why are some plasmids incompatible?
- Two plasmid species have same repl’cn control specificity
2. Random choice for replication from common plasmid pool
How to study plasmid replication control?
- E/m and 2-D gels
- Biochemistry
- Physiology (growth shifts, density shifts)
- Genetics; isolate mutants
- Molecular Genetics
what can Copy number mutants effect?
Quantitation and specificity of control.
what is the Basic Replicon?
- part of plasmid capable of independent autonomous replication at SAME COPY LEVEL as parental plasmid
- Fragment must carry all essential replication and copy control functions
- same incompatibility properties as parental plasmid
- Eg F factor 90kb
describe plasmid ColE1 (6.6kb)
- produces of colicin + immunity
- mobility functions
- oriV replication origin
- replication control functions
describe ColE1 replication
- RNAII produced upstream from oriV
- Forms DNA-RNA hybrid
- Hybrid cuts specific site by RNaseH to form 3’OH to prime DNA synthesis
- RNAI complementary to 5’ end of RNAII.
- binding of RNAI-RNAII helped by Rop
- Level of RNAI and/or Rop interferes with initiation of replication
how is ColE1 replication initiated?
- RNA-II changes conformation forms RNA/DNA duplex at oriV
2. RNA-II processed by RNaseH to form active primer for DNA replication by DNA Pol 1
how is replication controlled in ColE1?
- Interaction of RNA-I with RNA-II prevents RNA-II flipping into active conformation for formation of RNA/DNA duplex.
- Rop protein secondary control enhances efficiency of interaction between RNAI and RNAII prevent copy number drifting too high.
name 3 ColE1 replication mutants?
- Rep ts affects RNAII/DNA interaction & primer processing blocks oriV activation
- Cop affect 3’ end RNAI and folding reduce efficiency as repressor; copy # rises
- Inc map in RNAI/RNAII region create new Inc group and affect copy level.
what are ColE1 Inc- mutants?
- DOMINANT
- COMPATIBLE with wt ColE1
- Mutations map to RNA loops only
- Mutant plasmids have altered copy level
– GC>AT: increases copy level - Mutations affect incompatibility + copy level
what is ROP?
- Secondary control of copy number
- 63aa protein
- ColE1 Rop- recessive mutants have elevated copy number
whats the role of Rop?
- Rop protein accelerates RNA1-RNAII loop “kissing” interaction
- Binds to stems to aid positioning of complementary loops
- Sharpens “switch-OFF” of RNAII by RNAI
- Only if copy number too high
why do plasmids need Secondary Copy Controls?
- copy level too high disadvantageous/lethal to cell
2. copy level too low risk of plasmid-free cell at division
describe the plasmid F Fertility Factor of E.coli (100kB)
Basic replicon = 1.5kb EcoR1 fragment
carrying orin and RepA gene needed for initiation
describe Replication control of plasmids P1 + F
- RepA Positive activator of oriV
- CopB inhibits RepA transcription
- as plasmid enters cell no CopB so increased replication
- [CopB] inhibits RepA
- 2nd control by asRNA CopA.
what is DNA “handcuffing”?
- Plasmid replication control.
- At low concentrations of RepA;- monomeric protein interacts with oriV as initiator
At high concentrations of RepA;- monomer proteins bound to oriV/iterons of two molecules form dimers so “handcuffing” the two plasmid molecules and preventing further initiation
what factors contribute to stable plasmid maintenance?
- Decatenation (TopoIV) linked replication products
- Multimer resolution (P1 cre/lox : ColE1 cer/XerCD) – head/tail multimers by homologous recombin’
- Partitioning/Segregation (F Par, P1 Sop) – active distribution to daughter cells
- Addiction (R1 Hok/Sok) Post-segregational lethality
- Horizontal Transfer (F Tra) – conjugation and mobilisation
what is DECATENATION?
Product of DNA replication of circular dsDNAis linked circles
what is Multimer Resolution?
- Multimers arise by homologous recombination
- Rec-mediated Reduces No of segregation units
- Immediate loss problem for low copy plasmids
- Increase No oriV per unit
- Dimer catastrophe for multicopy random choice
plasmids, leads to eventual loss - Resolved by site-specific recombination system
encoded by plasmid - P1: cre recombinase @ lox site • ColE1: XerC/D @ cer site
what is Partitioning?
• Probability of a plasmid-free cell at division P0 = 2(1-n) where n = copy number
• Observed in vivo loss rate for plasmids = 10-6 to 10-7 per cell division
• Low copy plasmids have active systems F; SopC + SopA/B: site + proteins
P1; ParS + Par A/B: site + proteins
describe plasmid partitioning.
- ParB dimer binds to Par S site
- Nucleates binding of more parB dimers to form ParB filament between 2 plamids
- ParA (ATPase) regulates expression & polymerisation of ParB
- Filament pushes plasmid molecules apart
- Plasmids become anchored?
- ParB filament dissociates
