genet 270 lec 4- plasmids
What are plasmids?
xtrachromosomal DNA elements found in all types of bacteria; often multiple plasmids per cell
What are typical plasmid structures?
Usually covalently closed, circular dsDNA; sometimes linear or ssDNA. vary greatly in size and sometimes resemble the chromosome.
What do plasmids encode?
Genes (proteins, RNAs) that provide specific advantages (e.g., antibiotic resistance)
Why are plasmids important?
They aid bacterial adaptation & evolution and are powerful tools in molecular biology
How were plasmids originally named?
After the functions they encode (e.g., R plasmids = antibiotic resistance).
How are plasmids named now?
Standardized lab naming: “p” + creator initials + version (e.g., pUC19, pTR1)
What structural properties do plasmids usually have?
Covalently closed, circular dsDNA, negatively supercoiled
Why is supercoiling important in plasmids?
supercoil stress relieved by protein binding and used to facilitate replication and transcription
Why is plasmid purification possible?
Plasmids are small, allowing for selective precipitation of bacterial chromosome and other macromolecules
Outline plasmid purification steps:
Lyse cells with detergent & base.
Precipitate chromosome & other macromolecules with salt solution.
Bind soluble plasmid DNA to column → wash → elute
What is required for plasmid replication?
An origin of replication (oriV).
How do plasmids replicate independently?
They act as replicons — units of DNA with their own replication origin
They act as replicons — units of DNA with their own replication origin
Usually encode only 1 protein near oriV; other proteins are borrowed from the host
What is theta replication?
Common plasmid replication mechanism where replication starts at oriV using RNA primer that intiates DNAP replication, proceeds unidirectionally or bidirectionally around the plasmid
Which plasmids use theta replication?
ColE1, RK2, F, P1 (also the mechanism for bacterial chromosomes, though not called theta there)
What is rolling circle replication?
A replication mechanism producing ssDNA intermediates, later converted to dsDNA
What initiates rolling circle replication?
A nick introduced by Rep protein at ds oriV (DSO), creating a 3′-OH for DNA Pol III extension, displacing the parental strand. lagging strand synthesis occurs on displaced strand. produces long concatemers in which the original parental dna seq is repeated many times
How is ssDNA converted to dsDNA in RC replication?
1. rep protein binds at ds oriV @ palindrome
2. causes formation of hairpin cruciform structure
3.nicking; rep covalently bounds by Tyr to 5'-PO4
4.DNA pol II extends 3'OH
5. rep transfers 5' PO4 to 3'OH to create circular ssDNA
6. host RNAP makes primer at ss oriV
7. DNA pol III replicates around ssDNA circle
8. DNA pol I removes RNA primer, replaces with DNA, ligases seals
Where is rolling circle replication used?
By some phages, during bacterial conjugation, and in certain plasmids
What unusual products can RC replication form?
Long concatemers (multiple repeated plasmid units)
What is plasmid host range?
The range of bacterial species a plasmid can replicate in
What is a narrow host range plasmid? Give examples.
Replicates only in E. coli and close relatives (e.g., ColE1 plasmids: pBR322, pET, pUC)
What is a broad host range plasmid? Give examples.
Replicates in many Gram negatives and some Gram positives (e.g., RK2/RSF1010 derivatives such as pBBR1MCS)
How do broad host range plasmids achieve versatility?
Encode all proteins for replication (not dependent on host) and use promoter/translation (gene expression sequences) signals recognized by wide variety of machinery (promoters, translation, intiation sites)
What is required to determine host range experimentally?
Method to introduce plasmid into hosts + selectable marker trait (presentable phenotype encoded by plasmid to find in bacteria)
What is plasmid copy number?
The number of copies of a plasmid per cell, tightly controlled
What are relaxed plasmids?
High copy number plasmids; only regulate replication once numbers get very high
What are stringent plasmids?
Low copy number plasmids (as low as 1 per cell); replication must be tightly regulated
What is plasmid incompatibility?
Inability of two plasmids to coexist stably in the same cell
When does incompatibility occur?
Plasmids share the same replication control mechanism → not distinguished during replication → one lost randomly.
Plasmids share the same partitioning system → not distinguished during segregation → uneven distribution → one lost
What is an Inc group? How can Inc group be determined?
A classification for plasmids that cannot coexist in the same cell (same incompatibility group).By testing if a plasmid is cured from strains already containing a plasmid of a known Inc group- must have easily measured trait so can detect plasmid presence.
What are the 3 major plasmid replication control mechanisms?
sRNA interference with primer processing (ColE1-type).
Rep protein regulation via complementary RNA (R1-type).
Rep protein autoregulation & plasmid coupling (iterons/handcuffing) (pSC101-type)
Mechanism I – ColE1-type
Q: How does ColE1 replication control work?
RNA II normally forms replication primer. As plasmid copy number rises, sRNA RNA I accumulates → binds RNA II → prevents priming of replication
Mechanism II – R1-type
Q: How does R1 replication control work?
Rep protein required at oriV to seperate strands prior to replication. Rising copy number → sRNA accumulates --> CopA sRNA inhibits RepA translation, CopB repressor shuts off PrepA transcription
mechanism II- What happens right after plasmid entry into a cell?
Replication occurs until copy number is controlled by CopA/CopB feedback. CopB repressor made and shuts off PrepA and then copA RNA made and binds repA transcript to inhibit translation.
Mechanism III – pSC101-type
Q: How does iteron/handcuffing control work?
Rep protein at high levels binds its own promoter (autorepression) and iteron sites on plasmids → plasmids are coupled together (“handcuffing”) to block replication
Why is partitioning needed?
Low-copy plasmids must ensure each daughter cell inherits at least one copy
Which proteins carry out partitioning?
Par proteins (similar to bac chromosome segregation Par systems)
How does R1-type partitioning work? (search and capture)
ParR binds centromere-like parC site.
ParM-ATP polymerizes (actin-like filament) under ParR:parC.
Filament pushes plasmids apart.
ATP hydrolysis → depolymerization resets system
How does P1/F-type partitioning work?
ParA-ATP polymerizes along randomly on nucleoid DNA.
ParB binds parS site on plasmid.
ParB-parSattracted to ParA-ATP and stimulates ATP hydrolysis and depolymeriztion of ParA.
Depolymerization of parA, pulls plasmid and parB-parS across chromosome
Compare R1 vs P1/F partitioning:
R1-type = ParM pushes plasmids apart via actin-like filament polymerization.
P1/F-type = ParA pulls plasmid via depolymerization on nucleoid
Compare R1 vs P1/F: centromere, dna binding protein, actin like, movement.
R1; par C, Par R, ParM-ATP (at midpoint), pushes DNA apart- filament polymerizes to move DNA
P1/F: ParS, ParB, ParA-ATP (on nucleoid), pulls DNA apart- filament depolymerizes to move DNA
How do plasmids prevent being lost from cells?
Partitioning systems.
Site-specific recombinases to resolve plasmid multimers.
Toxin-antitoxin systems (cell dies if plasmid lost).
how have we exploited our knowledge of plasmids for molecular biology
plasmid cloning vectors