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Microbial Genomics > Plasmid Replication > Flashcards

Plasmid Replication Flashcards

1
Q

General Properties of plasmids

A
  1. Replicons with characteristic copy level maintenance functions
  2. Extrachromosomal some interact with chromosome.
  3. Small 1.5 – 200kb + replicate v quickly (max 2-3 min)
  4. Non-essential for viability of host cell can confer selective advantage on host (Abr)
  5. May be transmissible between cells - by conjugation or mobilisation
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2
Q

what are the two Replication modes of plasmids?

A
  1. democratic All replicated once only

2. random choice mixture are replicated.

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3
Q

whats Plasmid Incompatibility?

A

inability of two plasmid species to coexist

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4
Q

why are some plasmids incompatible?

A
  1. Two plasmid species have same repl’cn control specificity

2. Random choice for replication from common plasmid pool

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5
Q

How to study plasmid replication control?

A
  1. E/m and 2-D gels
  2. Biochemistry
  3. Physiology (growth shifts, density shifts)
  4. Genetics; isolate mutants
  5. Molecular Genetics
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6
Q

what can Copy number mutants effect?

A

Quantitation and specificity of control.

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7
Q

what is the Basic Replicon?

A
  1. part of plasmid capable of independent autonomous replication at SAME COPY LEVEL as parental plasmid
  2. Fragment must carry all essential replication and copy control functions
  3. same incompatibility properties as parental plasmid
  4. Eg F factor 90kb
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8
Q

describe plasmid ColE1 (6.6kb)

A
  1. produces of colicin + immunity
  2. mobility functions
  3. oriV replication origin
  4. replication control functions
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9
Q

describe ColE1 replication

A
  1. RNAII produced upstream from oriV
  2. Forms DNA-RNA hybrid
  3. Hybrid cuts specific site by RNaseH to form 3’OH to prime DNA synthesis
  4. RNAI complementary to 5’ end of RNAII.
  5. binding of RNAI-RNAII helped by Rop
  6. Level of RNAI and/or Rop interferes with initiation of replication
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10
Q

how is ColE1 replication initiated?

A
  1. 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

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11
Q

how is replication controlled in ColE1?

A
  1. Interaction of RNA-I with RNA-II prevents RNA-II flipping into active conformation for formation of RNA/DNA duplex.
  2. Rop protein secondary control enhances efficiency of interaction between RNAI and RNAII prevent copy number drifting too high.
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12
Q

name 3 ColE1 replication mutants?

A
  1. Rep ts affects RNAII/DNA interaction & primer processing blocks oriV activation
  2. Cop affect 3’ end RNAI and folding reduce efficiency as repressor; copy # rises
  3. Inc map in RNAI/RNAII region create new Inc group and affect copy level.
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13
Q

what are ColE1 Inc- mutants?

A
  1. DOMINANT
  2. COMPATIBLE with wt ColE1
  3. Mutations map to RNA loops only
  4. Mutant plasmids have altered copy level
    – GC>AT: increases copy level
  5. Mutations affect incompatibility + copy level
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14
Q

what is ROP?

A
  1. Secondary control of copy number
  2. 63aa protein
  3. ColE1 Rop- recessive mutants have elevated copy number
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15
Q

whats the role of Rop?

A
  1. Rop protein accelerates RNA1-RNAII loop “kissing” interaction
  2. Binds to stems to aid positioning of complementary loops
  3. Sharpens “switch-OFF” of RNAII by RNAI
  4. Only if copy number too high
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16
Q

why do plasmids need Secondary Copy Controls?

A
  1. copy level too high disadvantageous/lethal to cell

2. copy level too low risk of plasmid-free cell at division

17
Q

describe the plasmid F Fertility Factor of E.coli (100kB)

A

Basic replicon = 1.5kb EcoR1 fragment

carrying orin and RepA gene needed for initiation

18
Q

describe Replication control of plasmids P1 + F

A
  1. RepA Positive activator of oriV
  2. CopB inhibits RepA transcription
  3. as plasmid enters cell no CopB so increased replication
  4. [CopB] inhibits RepA
  5. 2nd control by asRNA CopA.
19
Q

what is DNA “handcuffing”?

A
  1. Plasmid replication control.
  2. 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
20
Q

what factors contribute to stable plasmid maintenance?

A
  1. Decatenation (TopoIV) linked replication products
  2. Multimer resolution (P1 cre/lox : ColE1 cer/XerCD) – head/tail multimers by homologous recombin’
  3. Partitioning/Segregation (F Par, P1 Sop) – active distribution to daughter cells
  4. Addiction (R1 Hok/Sok) Post-segregational lethality
  5. Horizontal Transfer (F Tra) – conjugation and mobilisation
21
Q

what is DECATENATION?

A

Product of DNA replication of circular dsDNAis linked circles

22
Q

what is Multimer Resolution?

A
  1. Multimers arise by homologous recombination
  2. Rec-mediated Reduces No of segregation units
  3. Immediate loss problem for low copy plasmids
  4. Increase No oriV per unit
  5. Dimer catastrophe for multicopy random choice
    plasmids, leads to eventual loss
  6. Resolved by site-specific recombination system
    encoded by plasmid
  7. P1: cre recombinase @ lox site • ColE1: XerC/D @ cer site
23
Q

what is Partitioning?

A

• 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

24
Q

describe plasmid partitioning.

A
  1. ParB dimer binds to Par S site
  2. Nucleates binding of more parB dimers to form ParB filament between 2 plamids
  3. ParA (ATPase) regulates expression & polymerisation of ParB
  4. Filament pushes plasmid molecules apart
  5. Plasmids become anchored?
  6. ParB filament dissociates
25
Q

what is Post-segregation lethality?

A

Host cell dies if plasmid is lost
• Two component systems - long half-life toxin
- short half-life antidote
• Plasmid R1
hok – protein causes respiratory failure
sok – antisense RNA inhibitor of hok expression
• Plasmid F
ccdB – inhibitor of DNA Gyrase ccdA – inhibitor of ccdB

26
Q

name all the maintenance systems

A
  1. Replication/copy control (essential)
  2. Monomerisation (multimer resolution, decatenation)
  3. Partitioning/segregation
  4. Post-segregation lethality
  5. Horizontal transfer (conjugation/mobilisation)

Microbial Genomics (7 decks)

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